POMC neurons control fertility through differential signaling of MC4R in Kisspeptin neurons.

Inactivating mutations in the melanocortin 4 receptor (MC4R) gene cause monogenic obesity. Interestingly, female patients also display various degrees of reproductive disorders, in line with the subfertile phenotype of MC4RKO female mice. However, the cellular mechanisms by which MC4R regulates reproduction are unknown. Kiss1 neurons directly stimulate gonadotropin-releasing hormone (GnRH) release through two distinct populations; the Kiss1ARH neurons, controlling GnRH pulses, and the sexually dimorphic Kiss1AVPV/PeN neurons controlling the preovulatory LH surge. Here, we show that Mc4r expressed in Kiss1 neurons is required for fertility in females. In vivo, deletion of Mc4r from Kiss1 neurons in female mice replicates the reproductive impairments of MC4RKO mice without inducing obesity. Conversely, reinsertion of Mc4r in Kiss1 neurons of MC4R null mice restores estrous cyclicity and LH pulsatility without reducing their obese phenotype. In vitro, we dissect the specific action of MC4R on Kiss1ARH vs Kiss1AVPV/PeN neurons and show that MC4R activation excites Kiss1ARH neurons through direct synaptic actions. In contrast, Kiss1AVPV/PeN neurons are normally inhibited by MC4R activation except under elevated estradiol levels, thus facilitating the activation of Kiss1AVPV/PeN neurons to induce the LH surge driving ovulation in females. Our findings demonstrate that POMCARH neurons acting through MC4R, directly regulate reproductive function in females by stimulating the "pulse generator" activity of Kiss1ARH neurons and restricting the activation of Kiss1AVPV/PeN neurons to the time of the estradiol-dependent LH surge, and thus unveil a novel pathway of the metabolic regulation of fertility by the melanocortin system.

CRISPR/SaCas9 mutagenesis of stromal interaction molecule 1 in proopiomelanocortin neurons increases glutamatergic excitability and protects against diet-induced obesity.

OBJECTIVE: Proopiomelanocortin (POMC) neurons are the key anorexigenic hypothalamic neuron for integrating metabolic cues to generate the appropriate output for maintaining energy homeostasis and express the requisite channels as a perfect synaptic integrator in this role. Similar to the metabolic hormones leptin and insulin, glutamate also excites POMC neurons via group I metabotropic glutamate receptors (mGluR1 and 5, mGluR1/5) that activate Transient Receptor Potential Canonical (TRPC 5) Channels to cause depolarization. A key modulator of TRPC 5 channel activity is stromal interaction molecule 1 (STIM1), which is involved in recruitment of TRPC 5 channels from receptor-operated to store-operated calcium entry following depletion of calcium from the endoplasmic reticulum. METHODS: We used a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas) and a single guide RNA (sgRNA) to mutate Stim1 in POMCCre neurons in male mice, verified by qPCR of Stim1 mRNA expression in single POMC neurons. Whole-cell patch clamp experiments were conducted to validate the effects of Stim1 mutagenesis. Body weight and food intake were measured in male mice to assess disruptions in energy balance. RESULTS: Reduced Stim1 expression augmented the efficacy of the mGluR1/5 agonist 3, 5-Dihydroxyphenylglycine (DHPG) to depolarize POMC neurons via a Gαq-coupled signaling pathway, which is an essential part of excitatory glutamatergic input in regulating energy homeostasis. The TRPC 5 channel blockers HC070 and Pico145 antagonized the excitatory effects of DHPG. As proof of principle, mutagenesis of Stim1 in POMC neurons reduced food intake, attenuated weight gain, reduced body fat and fat pad mass in mice fed a high fat diet. CONCLUSIONS: Using CRISPR technology we have uncovered a critical role of STIM1 in modulating glutamatergic activation of TRPC 5 channels in POMC neurons, which ultimately is important for maintaining energy balance.

Estrogenic regulation of reproduction and energy homeostasis by a triumvirate of hypothalamic arcuate neurons.

Pregnancy is energetically demanding and therefore, by necessity, reproduction and energy balance are inextricably linked. With insufficient or excessive energy stores a female is liable to suffer complications during pregnancy or produce unhealthy offspring. Gonadotropin-releasing hormone neurons are responsible for initiating both the pulsatile and subsequent surge release of luteinizing hormone to control ovulation. Meticulous work has identified two hypothalamic populations of kisspeptin (Kiss1) neurons that are critical for this pattern of release. The involvement of the hypothalamus is unsurprising because its quintessential function is to couple the endocrine and nervous systems, coordinating energy balance and reproduction. Estrogens, more specifically 17ß-estradiol (E2 ), orchestrate the activity of a triumvirate of hypothalamic neurons within the arcuate nucleus (ARH) that govern the physiological underpinnings of these behavioral dynamics. Arising from a common progenitor pool, these cells differentiate into ARH kisspeptin, pro-opiomelanocortin (POMC), and agouti related peptide/neuropeptide Y (AgRP) neurons. Although the excitability of all these subpopulations is subject to genomic and rapid estrogenic regulation, Kiss1 neurons are the most sensitive, reflecting their integral function in female fertility. Based on the premise that E2 coordinates autonomic functions around reproduction, we review recent findings on how Kiss1 neurons interact with gonadotropin-releasing hormone, AgRP and POMC neurons, as well as how the rapid membrane-initiated and intracellular signaling cascades activated by E2 in these neurons are critical for control of homeostatic functions supporting reproduction. In particular, we highlight how Kiss1 and POMC neurons conspire to inhibit AgRP neurons and diminish food motivation in service of reproductive success.

Deletion of Stim1 in Hypothalamic Arcuate Nucleus Kiss1 Neurons Potentiates Synchronous GCaMP Activity and Protects against Diet-Induced Obesity.

Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. High-frequency firing of hypothalamic arcuate Kiss1 (Kiss1ARH) neurons releases kisspeptin into the median eminence, and neurokinin B (NKB) and dynorphin onto neighboring Kiss1ARH neurons to generate a slow EPSP mediated by TRPC5 channels that entrains intermittent, synchronous firing of Kiss1ARH neurons. High-frequency optogenetic stimulation of Kiss1ARH neurons also releases glutamate to excite the anorexigenic proopiomelanocortin (POMC) neurons and inhibit the orexigenic neuropeptide Y/agouti-related peptide (AgRP) neurons via metabotropic glutamate receptors. At the molecular level, the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1) is critically involved in the regulation of neuronal Ca2+ signaling and neuronal excitability through its interaction with plasma membrane (PM) calcium (e.g., TRPC) channels. Therefore, we hypothesized that deletion of Stim1 in Kiss1ARH neurons would increase neuronal excitability and their synchronous firing, which ultimately would affect energy homeostasis. Using optogenetics in combination with whole-cell recording and GCaMP6 imaging in slices, we discovered that deletion of Stim1 in Kiss1 neurons significantly increased the amplitude and duration of the slow EPSP and augmented synchronous [Ca2+]i oscillations in Kiss1ARH neurons. Deletion of Stim1 in Kiss1ARH neurons amplified the actions of NKB and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD). Therefore, STIM1 appears to play a critical role in regulating synchronous firing of Kiss1ARH neurons, which ultimately affects the coordination between energy homeostasis and reproduction.SIGNIFICANCE STATEMENT Hypothalamic arcuate kisspeptin (Kiss1ARH) neurons are essential for stimulating the pulsatile release of gonadotropin-releasing hormone (GnRH) and maintaining fertility. However, Kiss1ARH neurons appear to be a key player in coordinating energy balance with reproduction. The regulation of calcium channels and hence calcium signaling is critically dependent on the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1), which interacts with the plasma membrane (PM) calcium channels. We have conditionally deleted Stim1 in Kiss1ARH neurons and found that it significantly increased the excitability of Kiss1ARH neurons and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD).

Arcuate and Preoptic Kisspeptin Neurons Exhibit Differential Projections to Hypothalamic Nuclei and Exert Opposite Postsynaptic Effects on Hypothalamic Paraventricular and Dorsomedial Nuclei in the Female Mouse.

Kisspeptin (Kiss1) neurons provide indispensable excitatory input to gonadotropin-releasing hormone (GnRH) neurons, which is important for the coordinated release of gonadotropins, estrous cyclicity and ovulation. However, Kiss1 neurons also send projections to many other brain regions within and outside the hypothalamus. Two different populations of Kiss1 neurons, one in the arcuate nucleus (Kiss1ARH) and another in the anteroventral periventricular nucleus (AVPV) and periventricular nucleus (PeN; Kiss1AVPV/PeN) of the hypothalamus are differentially regulated by ovarian steroids, and are believed to form direct contacts with GnRH neurons as well as other neurons. To investigate the projection fields from Kiss1AVPV/PeN and Kiss1ARH neurons in female mice, we used anterograde projection analysis, and channelrhodopsin-assisted circuit mapping (CRACM) to explore their functional input to select target neurons within the paraventricular (PVH) and dorsomedial (DMH) hypothalamus, key preautonomic nuclei. Cre-dependent viral (AAV1-DIO-ChR2 mCherry) vectors were injected into the brain to label the two Kiss1 neuronal populations. Immunocytochemistry (ICC) for mCherry and neuropeptides combined with confocal microscopy was used to determine the projection-fields of both Kiss1 neuronal groups. Whole-cell electrophysiology and optogenetics were used to elucidate the functional input to the PVH and DMH. Our analysis revealed many common but also several clearly separate projection fields between the two different populations of Kiss1 neurons. In addition, optogenetic stimulation of Kiss1 projections to PVH prodynorphin, Vglut2 and DMH CART-expressing neurons, revealed excitatory glutamatergic input from Kiss1ARH neurons and inhibitory GABAergic input from Kiss1AVPV/PeN neurons. Therefore, these steroid-sensitive Kiss1 neuronal groups can differentially control the excitability of target neurons to coordinate autonomic functions with reproduction.

CRISPR knockdown of Kcnq3 attenuates the M-current and increases excitability of NPY/AgRP neurons to alter energy balance.

OBJECTIVE: Arcuate nucleus neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior. The M-current, a subthreshold non-inactivating potassium current, plays a critical role in regulating NPY/AgRP neuronal excitability. Fasting decreases while 17ß-estradiol increases the M-current by regulating the mRNA expression of Kcnq2, 3, and 5 (Kv7.2, 3, and 5) channel subunits. Incorporating KCNQ3 into heteromeric channels has been considered essential to generate a robust M-current. Therefore, we investigated the behavioral and physiological effects of selective Kcnq3 deletion from NPY/AgRP neurons. METHODS: We used a single adeno-associated viral vector containing a recombinase-dependent Staphylococcus aureus Cas9 with a single-guide RNA to selectively delete Kcnq3 in NPY/AgRP neurons. Single-cell quantitative measurements of mRNA expression and whole-cell patch clamp experiments were conducted to validate the selective knockdown. Body weight, food intake, and locomotor activity were measured in male mice to assess disruptions in energy balance. RESULTS: The virus reduced the expression of Kcnq3 mRNA without affecting Kcnq2 or Kcnq5. The M-current was attenuated, causing NPY/AgRP neurons to be more depolarized, exhibit a higher input resistance, and require less depolarizing current to fire action potentials, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it significantly reduced the locomotor activity as measured by open-field testing. Control mice on a high-fat diet exhibited an enhanced M-current and increased Kcnq2 and Kcnq3 expression, but the M-current remained significantly attenuated in KCNQ3 knockdown animals. CONCLUSIONS: The M-current plays a critical role in modulating the intrinsic excitability of NPY/AgRP neurons that is essential for maintaining energy homeostasis.

Membrane and nuclear initiated estrogenic regulation of homeostasis.

Reproduction and energy balance are inextricably linked in order to optimize the evolutionary fitness of an organism. With insufficient or excessive energy stores a female is liable to suffer complications during pregnancy and produce unhealthy or obesity-prone offspring. The quintessential function of the hypothalamus is to act as a bridge between the endocrine and nervous systems, coordinating fertility and autonomic functions. Across the female reproductive cycle various motivations wax and wane, following levels of ovarian hormones. Estrogens, more specifically 17ß-estradiol (E2), coordinate a triumvirate of hypothalamic neurons within the arcuate nucleus (ARH) that govern the physiological underpinnings of these behavioral dynamics. Arising from a common progenitor pool of cells, this triumvirate is composed of the kisspeptin (Kiss1ARH), proopiomelanocortin (POMC), and neuropeptide Y/agouti-related peptide (AgRP) neurons. Although the excitability of these neuronal subpopulations is subject to genomic and rapid estrogenic regulation, kisspeptin neurons are the most sensitive, reflecting their integral function in female fertility. Based on the premise that E2 coordinates autonomic functions around reproduction, we will review the recent findings on the synaptic interactions between Kiss1, AgRP and POMC neurons and how the rapid membrane-initiated and intracellular signaling cascades activated by E2 in these neurons are critical for control of homeostatic functions supporting reproduction.

Estradiol Drives the Anorexigenic Activity of Proopiomelanocortin Neurons in Female Mice.

Energy balance is regulated by anorexigenic proopiomelanocortin (POMC) and orexigenic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons of the hypothalamic arcuate nucleus. POMC neurons make extensive projections and are thought to release both amino acid and peptide neurotransmitters. However, whether they communicate directly with NPY/AgRP neurons is debated. Initially, using single-cell RT-PCR, we determined that mouse POMCeGFP neurons express Slc17a6 (Vglut2) and Slc18a2 (Vmat2), but not Slc31a1 (Vgat) mRNA, suggesting glutamate and non-canonical GABA release. Quantitative (q)RT-PCR of POMCeGFP cells revealed that Vglut2 and Vmat2 expression was significantly increased in E2- versus oil-treated, ovariectomized (OVX) female mice. Since 17ß-estradiol (E2) is anorexigenic, we hypothesized that an underlying mechanism is enhancement of POMC signaling. Therefore, we optogenetically stimulated POMC neurons in hypothalamic slices to examine evoked release of neurotransmitters onto NPY/AgRP neurons. Using brief light pulses, we primarily observed glutamatergic currents and, based on the paired pulse ratio (PPR), determined that release probability was higher in E2- versus oil-treated, OVX female, congruent with increased Vlgut2 expression. Moreover, bath perfusion of the Gq-coupled membrane estrogen receptor (ER) agonist STX recapitulated the effects of E2 treatment. In addition, high-frequency (20 Hz) stimulation generated a slow outward current that reversed near Ek+ and was antagonized by naloxone, indicative of ß-endorphin release. Furthermore, individual NPY/AgRP neurons were found to express Oprm1, the transcript for µ-opioid receptor, and DAMGO, a selective agonist, elicited an outward current. Therefore, POMC excitability and neurotransmission are enhanced by E2, which would facilitate decreased food consumption through marked inhibition of NPY/AgRP neurons.

Estrogenic-dependent glutamatergic neurotransmission from kisspeptin neurons governs feeding circuits in females.

The neuropeptides tachykinin2 (Tac2) and kisspeptin (Kiss1) in hypothalamic arcuate nucleus Kiss1 (Kiss1ARH) neurons are essential for pulsatile release of GnRH and reproduction. Since 17ß-estradiol (E2) decreases Kiss1 and Tac2 mRNA expression in Kiss1ARH neurons, the role of Kiss1ARH neurons during E2-driven anorexigenic states and their coordination of POMC and NPY/AgRP feeding circuits have been largely ignored. Presently, we show that E2 augmented the excitability of Kiss1ARH neurons by amplifying Cacna1g, Hcn1 and Hcn2 mRNA expression and T-type calcium and h-currents. E2 increased Slc17a6 mRNA expression and glutamatergic synaptic input to arcuate neurons, which excited POMC and inhibited NPY/AgRP neurons via metabotropic receptors. Deleting Slc17a6 in Kiss1 neurons eliminated glutamate release and led to conditioned place preference for sucrose in E2-treated KO female mice. Therefore, the E2-driven increase in Kiss1 neuronal excitability and glutamate neurotransmission may play a key role in governing the motivational drive for palatable food in females.

Bistratified starburst amacrine cells in Sox2 conditional knockout mouse retina display ON and OFF responses.

Cell-intrinsic factors, in conjunction with environmental signals, guide migration, differentiation, and connectivity during early development of neuronal circuits. Within the retina, inhibitory starburst amacrine cells (SBACs) comprise ON types with somas in the ganglion cell layer (GCL) and dendrites stratifying narrowly in the inner half of the inner plexiform layer (IPL) and OFF types with somas in the inner nuclear layer (INL) and dendrites stratifying narrowly in the outer half of the IPL. The transcription factor Sox2 is crucial to this subtype specification. Without Sox2, many ON-type SBACs destined for the GCL settle in the INL while many that reach the GCL develop bistratified dendritic arbors. This study asked whether ON-type SBACs in Sox2-conditional knockout retinas exhibit selective connectivity only with ON-type bipolar cells or their bistratified morphology allows them to connect to both ON and OFF bipolar cells. Physiological data demonstrate that these cells receive ON and OFF excitatory inputs, indicating that the ectopically stratified dendrites make functional synapses with bipolar cells. The excitatory inputs were smaller and more transient in Sox2-conditional knockout compared with wild type; however, inhibitory inputs appeared largely unchanged. Thus dendritic stratification, rather than cellular identification, may be the major factor that determines ON vs. OFF connectivity. NEW & NOTEWORTHY Conditional knockout of the transcription factor Sox2 during early embryogenesis converts a monostratifying starburst amacrine cell into a bistratifying starburst cell. Here we show that these bistratifying starburst amacrine cells form functional synaptic connections with both ON and OFF bipolar cells. This suggests that normal ON vs. OFF starburst connectivity may not require distinct molecular specification. Proximity alone may be sufficient to allow formation of functional synapses.

Diverse actions of estradiol on anorexigenic and orexigenic hypothalamic arcuate neurons.

Contribution to Special Issue on Fast effects of steroids. There is now compelling evidence for membrane-associated estrogen receptors in hypothalamic neurons that are critical for the hypothalamic control of homeostatic functions. It has been known for some time that estradiol (E2) can rapidly alter hypothalamic neuronal activity within seconds, indicating that some cellular effects can occur via membrane initiated events. However, our understanding of how E2 signals via membrane-associated receptors and how these signals impact physiological functions is only just emerging. Thus, E2 can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell excitability and even gene transcription in hypothalamic neurons. One population of hypothalamic neurons, the anorexigenic proopiomelanocortin (POMC) neurons, has long been considered to be a target of E2's actions based on gene (Pomc) expression studies. However, we now know that E2 can rapidly alter POMC neuronal activity within seconds and activate several intracellular signaling cascades that ultimately affect gene expression, actions which are critical for maintaining sensitivity to insulin in metabolically stressed states. E2 also affects the orexigenic Neuropeptide Y/Agouti-related Peptide (NPY/AgRP) neurons in similarly rapid but antagonistic manner. Therefore, this review will summarize our current state of knowledge of how E2 signals via rapid membrane-initiated and intracellular signaling cascades in POMC and NPY/AgRP neurons to regulate energy homeostasis.

Different AMPA receptor subtypes mediate the distinct kinetic components of a biphasic EPSC in hippocampal interneurons.

CA1 hippocampal interneurons at the border between stratum radiatum (SR) and stratum lacunosum-moleculare (SLM) have AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents (EPSCs) that consist of two distinct phases: a typical fast component (FC), and a highly unusual slow component (SC) that persists for hundreds of milliseconds. To determine whether these kinetically distinct components of the EPSC are mediated by distinct AMPAR subpopulations, we examined the relative contributions of GluA2-containing and-lacking AMPARs to the SC. GluA2-containing AMPARs mediated the majority of the FC whereas GluA2-lacking AMPARs preferentially generated the SC. When glutamate uptake through the glial glutamate transporter excitatory amino acid transporter (EAAT1) was inhibited, spill over-mediated AMPAR activation recruited an even slower third kinetic component that persisted for several seconds; however, this spillover-mediated current was mediated predominantly by GluA2-containing AMPARs and therefore was clearly distinct from the SC when uptake is intact. Thus, different AMPAR subpopulations that vary in GluA2 content mediate the distinct components of the AMPAR EPSC. The SC is developmentally downregulated in mice, declining after the second postnatal week. This downregulation affects both GluA2-containing and GluA2-lacking AMPARs mediating the SC, and is not accompanied by developmental changes in the GluA2 content of AMPARs underlying the FC. Thus, the downregulation of the SC appears to be independent of synaptic GluA2 expression, suggesting the involvement of another AMPAR subunit or an auxiliary protein. Our results therefore identify GluA2-dependent and GluA2-independent determinants of the SC: GluA2-lacking AMPARs preferentially contribute to the SC, while the developmental downregulation of the SC is independent of GluA2 content.

Localization of CB1 cannabinoid receptor mRNA in the brain of the chick (Gallus domesticus).

The cannabinoid receptor one (CB1) is prevalent in the brains of many species. Receptor binding, in situ hybridization and immunohistochemical surveys have described the distribution of this receptor in a limited number of species. The current study used in situ hybridization to examine the expression of CB1 mRNA in the chick brain, a non-mammalian vertebrate. The results were compared to the observed patterns of expression for CB1 mRNA, protein, and agonist binding that have been reported for other avian species and mammals. Importantly, since CB1 receptors are typically located on neuronal terminals, comparison of the somatic mRNA expression with previously reported descriptions of the location of functional receptors, allows speculation about the circuits that make use of these receptors. The expression pattern for CB1 mRNA appears to be highly conserved across species in key areas such as the cerebellum and portions of the forebrain. For example, high levels of expression were observed in the avian amygdala and hippocampus, areas which express high levels of CB1 in mammals. The avian substantia nigra and ventral tegmental area, however, showed specific labeling. This finding is in stark contrast to the high levels of receptor binding or CB1 protein, but not CB1 mRNA in these areas of the mammalian brain. Moderate labeling was also seen throughout the hyperpallium and mesopallium. Throughout the brain, a number of regions that are known to be involved in visual processing displayed high levels of expression. For example, the tectum also had strong mRNA expression within layers 9-11 of the stratum griseum et fibrosum superficale and stratum album centrale.

Oestrogen and weight loss decrease isoproterenol-induced Fos immunoreactivity and angiotensin type 1 mRNA in the subfornical organ of female rats.

Studies from our laboratory and others show that oestrogen reduces angiotensin II (Ang II)-induced water intake by ovariectomized rats. Elimination of endogenous oestrogen by ovariectomy causes weight gain that can be reversed or prevented by oestrogen replacement. Changes in body weight modify cardiovascular responses to Ang II but whether such changes have similar effects on central and behavioural responses to Ang II is unknown. The goal of this study was to evaluate the contributions of oestrogen and weight loss to isoproterenol (isoprenaline; Iso)-induced Fos immunoreactivity (IR) and to angiotensin type 1 (AT1) receptor mRNA in forebrain regions implicated in the control of fluid balance. Isoproterenol significantly increased Fos IR in the hypothalamic paraventricular and supraoptic nuclei, the subfornical organ (SFO), and the organum vasculosum of the lamina terminalis, but had no effect on AT1 mRNA expression. However, both Iso-induced Fos IR and the AT1 mRNA were attenuated in the SFO of the oestrogen and weight loss groups compared with that of the control group. Consequently, we examined the effect of weight loss on Iso-induced water intake and plasma renin activity (PRA) and found that weight loss decreased water intake after Iso, but had no effect on PRA. Thus, we propose that weight loss decreases Ang II-elicited water intake in the female rat by down-regulating the expression of the AT1 receptor.

Differential effects of antipsychotics on haloperidol-induced vacuous chewing movements and subcortical gene expression in the rat.

The behavioral and neurochemical effects of switching from typical to atypical medications have not been evaluated in the rodent models of tardive dyskinesia. Thus, we treated rats with haloperidol-decanoate for 12 weeks, and assessed the effects of additional treatment with olanzapine, haloperidol, clozapine, or vehicle on vacuous chewing movements and expression of transcripts for dopamine receptors, tyrosine hydroxylase, delta-opioid receptor, prodynorphin, preproenkephalin, glutamic acid decarboxylase-65 (glutamic acid decarboxylase (GAD)-65) and GAD-67 and N-methyl-D-aspartate (NMDA) receptor subunits in the striatum and its efferent pathways. Haloperidol-decanoate induced vacuous chewing movements extinguished following an additional 4 weeks of treatment with vehicle, olanzapine or haloperidol, but not clozapine. Post-treatment, vacuous chewing movements in the clozapine group were significantly higher than the vehicle, olanzapine and haloperidol groups. GAD-67 mRNA expression in the globus pallidus was decreased following additional treatment with olanzapine or haloperidol, but not clozapine. Changes in expression of other transcripts were not detected. These findings demonstrate important differences in the effects of typical and atypical antipsychotics on chronic vacuous chewing movements.