Wireless stimulation of the subthalamic nucleus with nanoparticles modulates key monoaminergic systems similar to contemporary deep brain stimulation.

BACKGROUND: Deep brain stimulation (DBS) is commonly used to alleviate motor symptoms in several movement disorders. However, the procedure is invasive, and the technology has remained largely stagnant since its inception decades ago. Recently, we have shown that wireless nanoelectrodes may offer an alternative approach to conventional DBS. However, this method is still in its infancy, and more research is required to characterize its potential before it can be considered as an alternative to conventional DBS. OBJECTIVES: Herein, we aimed to investigate the effect of stimulation via magnetoelectric nanoelectrodes on primary neurotransmitter systems that have implications for DBS in movement disorders. METHODS: Mice were injected with either magnetoelectric nanoparticles (MENPs) or magnetostrictive nanoparticles (MSNPs, as a control) in the subthalamic nucleus (STN). Mice then underwent magnetic stimulation, and their motor behavior was assessed in the open field test. In addition, magnetic stimulation was applied before sacrifice and post-mortem brains were processed for immunohistochemistry (IHC) to assess the co-expression of c-Fos with either tyrosine hydroxylase (TH), tryptophan hydroxylase-2 (TPH2) or choline acetyltransferase (ChAT). RESULTS: Stimulated animals covered longer distances in the open field test when compared to controls. Moreover, we found a significant increase in c-Fos expression in the motor cortex (MC) and paraventricular region of the thalamus (PV-thalamus) after magnetoelectric stimulation. Stimulated animals showed fewer TPH2/c-Fos double-labeled cells in the dorsal raphe nucleus (DRN), as well as TH/c-Fos double-labeled cells in the ventral tegmental area (VTA), but not in the substantia nigra pars compacta (SNc). There was no significant difference in the number of ChAT/ c-Fos double-labeled cells in the pedunculopontine nucleus (PPN). CONCLUSIONS: Magnetoelectric DBS in mice enables selective modulation of deep brain areas and animal behavior. The measured behavioral responses are associated with changes in relevant neurotransmitter systems. These changes are somewhat similar to those observed in conventional DBS, suggesting that magnetoelectric DBS might be a suitable alternative.

Alterations in NMDAR-mediated signaling within the laterodorsal tegmental nucleus are associated with prenatal nicotine exposure.

Cigarette smoking during pregnancy has been clinically associated with a variety of poorbehavioral outcomes for the exposed individuals, including higher risks for drug abuse and development of attention/deficit-hyperactive disorder (ADHD). Experimental studies support the hypothesis that nicotine might contribute to these risks, since prenatal nicotine exposure (PNE) in rodents was associated with greater addiction liability, hyperactivity, social impairments and a wide range of emotional and cognitive deficits. Alterations of glutamate signaling within brain regions involved in behavioral circuits could contribute to these outcomes. The pontine laterodorsal tegmental nucleus (LDT) exerts cholinergic modulation within the ventral tegmental area, nucleus accumbens, and cortical-projecting thalamic centers and PNE-associated alterations in LDT glutamate signaling could impact cholinergic output to these LDT targets. We have previously demonstrated that PNE alters AMPA-mediated signaling within LDT neurons, and in the present investigation, we focused on changes of NMDA receptors (NMDARs) and presence of silent synapses as an indicator of metaplastic processes in LDT cells associated with PNE treatment. PNE was associated with a decreased functional presence of GluN2B NMDAR subunits in synapses of large, putatively cholinergic neurons, whereas an increased function of this subunit was detected in small, likely GABAergic cells. In addition, PNE was associated with functional alterations of extrasynaptic NMDARs in putative cholinergic neurons, suggestive of an increased presence of GluN3A-containing NMDARs. An increased number of silent synapses was exclusively seen in the small cells. When taken together, we hypothesize that NMDA-mediated signaling changes within LDT neurons following PNE treatment would result in reductions of excitatory cholinergic modulatory tone in target brain regions, which would be expected to contribute to the behavioral deficits found among these individuals.

Noradrenaline from Locus Coeruleus Neurons Acts on Pedunculo-Pontine Neurons to Prevent REM Sleep and Induces Its Loss-Associated Effects in Rats.

Normally, rapid eye movement sleep (REMS) does not appear during waking or non-REMS. Isolated, independent studies showed that elevated noradrenaline (NA) levels inhibit REMS and induce REMS loss-associated cytomolecular, cytomorphological, psychosomatic changes and associated symptoms. However, the source of NA and its target in the brain for REMS regulation and function in health and diseases remained to be confirmed in vivo. Using tyrosine hydroxylase (TH)-siRNA and virus-coated TH-shRNA in normal freely moving rats, we downregulated NA synthesis in locus coeruleus (LC) REM-OFF neurons in vivo. These TH-downregulated rats showed increased REMS, which was prevented by infusing NA into the pedunculo-pontine tegmentum (PPT), the site of REM-ON neurons, normal REMS returned after recovery. Moreover, unlike normal or control-siRNA- or shRNA-injected rats, upon REMS deprivation (REMSD) TH-downregulated rat brains did not show elevated Na-K ATPase (molecular changes) expression and activity. To the best of our knowledge, these are the first in vivo findings in an animal model confirming that NA from the LC REM-OFF neurons (1) acts on the PPT REM-ON neurons to prevent appearance of REMS, and (2) are responsible for inducing REMSD-associated molecular changes and symptoms. These observations clearly show neuro-physio-chemical mechanism of why normally REMS does not appear during waking. Also, that LC neurons are the primary source of NA, which in turn causes some, if not many, REMSD-associated symptoms and behavioral changes. The findings are proof-of-principle for the first time and hold potential to be exploited for confirmation toward treating REMS disorder and amelioration of REMS loss-associated symptoms in patients.

Intracellular mechanisms modulating gamma band activity in the pedunculopontine nucleus (PPN).

The pedunculopontine nucleus is a part of the reticular activating system, and is active during waking and REM sleep. Previous results showed that all PPN cells tested fired maximally at gamma frequencies when depolarized. This intrinsic membrane property was shown to be mediated by high-threshold N- and P/Q-type Ca(2+) channels. Recent studies show that the PPN contains three independent populations of neurons which can generate gamma band oscillations through only N-type channels, only P/Q-type channels, or both N- and P/Q-type channels. This study investigated the intracellular mechanisms modulating gamma band activity in each population of neurons. We performed in vitro patch-clamp recordings of PPN neurons from Sprague-Dawley rat pups, and applied 1-sec ramps to induce intrinsic membrane oscillations. Our results show that there are two pathways modulating gamma band activity in PPN neurons. We describe populations of neurons mediating gamma band activity through only N-type channels and the cAMP/PKA pathway (presumed "REM-on" neurons), through only P/Q-type channels and the CaMKII pathway (presumed "Wake-on" neurons), and a third population which can mediate gamma activity through both N-type channels and cAMP/PK and P/Q-type channels and CaMKII (presumed "Wake/REM-on" neurons). These novel results suggest that PPN gamma oscillations are modulated by two independent pathways related to different Ca(2+) channel types.

Pedunculopontine cell loss and protein aggregation direct microglia activation in parkinsonian rats.

We previously reported a loss of cholinergic neurons within the pedunculopontine tegmental nucleus (PPTg) in rats that had been intra-nigrally lesioned with the proteasomal inhibitor lactacystin, with levels of neuronal loss corresponding to that seen in the post-mortem pedunculopontine nucleus (PPN) of advanced Parkinson's disease (PD) patients. Here we reveal lower expression values of the acetylcholine synthesising enzyme, choline acetyltransferase, within the remaining PPTg cholinergic neurons of lesioned rats compared to sham controls. We further characterise this animal model entailing dopaminergic- and non-dopaminergic neurodegeneration by reporting on stereological counts of non-cholinergic neurons, to determine whether the toxin is neuro-type specific. Cell counts between lesioned and sham-lesioned rats were analysed in terms of the topological distribution pattern across the rostro-caudal extent of the PPTg. The study also reports somatic hypotrophy in the remaining non-cholinergic neurons, particularly on the side closest to the nigral lesion. The cytotoxicity affecting the PPTg in this rat model of PD involves overexpression and accumulation of alpha-synuclein (αSYN), affecting cholinergic and non-cholinergic neurons as well as microglia on the lesioned hemispheric side. We ascertained that microglia within the PPTg become fully activated due to the extensive neuronal damage and neuronal death resulting from a lactacystin nigral lesion, displaying a distinct rostro-caudal distribution profile which correlates with PPTg neuronal loss, with the added implication that lactacystin-induced αSYN aggregation might trigger neuronophagia for promoting PPTg cell loss. The data provide critical insights into the mechanisms underlying the lactacystin rat model of PD, for studying the PPTg in health and when modelling neurodegenerative disease.

c-Fos expression after deep brain stimulation of the pedunculopontine tegmental nucleus in the rat 6-hydroxydopamine Parkinson model.

Deep brain stimulation (DBS) is used to alleviate motor dysfunction in Parkinson's disease (PD). The pedunculopontine nucleus (PPN) may be a potential target for severe freezing and postural instability with 25 Hz stimulation being considered more effective than 130 Hz stimulation. Here we evaluated the expression of c-Fos after 25 Hz and 130 Hz DBS of the pedunculopontine tegmental nucleus (PPTg, i.e., the rodent equivalent to the human PPN) in the rat 6-hydroxydopamine (6-OHDA) PD model. Anaesthetized male Sprague Dawley rats with unilateral 6-OHDA-induced nigrostriatal lesions were stimulated with 25 Hz, 130 Hz, or 0 Hz sham-stimulation for 4h by electrodes implanted into the ipsilateral PPTg. Thereafter the distribution and number of neurons expressing the immediate early gene c-Fos, a marker for acute neuronal activity, was assessed. DBS of the PPTg induced strong ipsilateral c-Fos expression at the stimulation site, with 25 Hz having a more marked impact than 130 Hz. Additionally, c-Fos was strongly expressed in the central gray. In the dorsal part expression was stronger after 25 Hz stimulation, while in the medial and ventral part there was no difference between 25 Hz and 130 Hz stimulation. Expression in the basal ganglia was negligible. In the rat 6-OHDA PD model stimulation of the PPTg did not affect c-Fos expression in the basal ganglia, but had a strong impact on other functional circuitries. PPN stimulation in humans might therefore also have an impact on other systems than the motor system.

Therapeutic potential of nicotine for methamphetamine-induced impairment of sensorimotor gating: involvement of pallidotegmental neurons.

INTRODUCTION: We have previously found that a disruption to prepulse inhibiton (PPI) induced by methamphetamine (METH) is associated with impaired functioning of pallidotegmental neurons, which play a crucial role in PPI of the startle reflex, through the activation of gamma-aminobutyric acid type B receptors in pedunculopontine tegmental neurons in mice. OBJECTIVES: Here, we examined the effect of nicotine on METH-induced impairment of PPI of the startle reflex focusing on dysfunctional pallidotegmental neurons and the neural system. RESULTS: Nicotine (0.15-0.5 mg/kg) ameliorated the deficit in PPI induced by acute METH, and the ameliorating effect of nicotine was antagonized by nicotinic receptor antagonists such as methyllycaconitine and dihydro-beta-erythroidine. The acute METH-induced disruption of PPI was accompanied by suppression of c-Fos expression in the lateral globus pallidus (LGP) as well as its induction in the caudal pontine reticular nucleus (PnC) in mice subjected to the PPI test. Nicotine-induced amelioration of PPI deficits in METH-treated mice was accompanied by a reversal of the changes in c-Fos expression in both the LGP and PnC to the basal level. CONCLUSIONS: Nicotine is effective in ameliorating the impairment of PPI caused by METH, which may be associated with normalization of the pallidotegmental neurons.

Identification of cholinergic and non-cholinergic neurons in the pons expressing phosphorylated cyclic adenosine monophosphate response element-binding protein as a function of rapid eye movement sleep.

Recent studies have shown that in the pedunculopontine tegmental nucleus (PPT), increased neuronal activity and kainate receptor-mediated activation of intracellular protein kinase A (PKA) are important physiological and molecular steps for the generation of rapid eye movement (REM) sleep. In the present study performed on rats, phosphorylated cyclic AMP response element-binding protein (pCREB) immunostaining was used as a marker for increased intracellular PKA activation and as a reflection of increased neuronal activity. To identify whether activated cells were either cholinergic or noncholinergic, the PPT and laterodorsal tegmental nucleus (LDT) cells were immunostained for choline acetyltransferase (ChAT) in combination with pCREB or c-Fos. The results demonstrated that during high rapid eye movement sleep (HR, approximately 27%), significantly higher numbers of cells expressed pCREB and c-Fos in the PPT, of which 95% of pCREB-expressing cells were ChAT-positive. With HR, the numbers of pCREB-positive cells were also significantly higher in the medial pontine reticular formation (mPRF), pontine reticular nucleus oral (PnO), and dorsal subcoeruleus nucleus (SubCD) but very few in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). Conversely, with low rapid eye movement sleep (LR, approximately 2%), the numbers of pCREB expressing cells were very few in the PPT, mPRF, PnO, and SubCD but significantly higher in the LC and DRN. The results of regression analyses revealed significant positive relationships between the total percentages of REM sleep and numbers of ChAT+/pCREB+ (Rsqr=0.98) cells in the PPT and pCREB+ cells in the mPRF (Rsqr=0.88), PnO (Rsqr=0.87), and SubCD (Rsqr=0.84); whereas significantly negative relationships were associated with the pCREB+ cells in the LC (Rsqr=0.70) and DRN (Rsqr=0.60). These results provide evidence supporting the hypothesis that during REM sleep, the PPT cholinergic neurons are active, whereas the LC and DRN neurons are inactive. More importantly, the regression analysis indicated that pCREB activation in approximately 98% of PPT cholinergic neurons, was caused by REM sleep. Moreover the results indicate that during REM sleep, PPT intracellular PKA activation and a transcriptional cascade involving pCREB occur exclusively in the cholinergic neurons.

Effects of nicotine, methamphetamine and cocaine on extracellular levels of acetylcholine in the interpeduncular nucleus of rats.

There is increasing evidence that the cholinergic habenulo-interpeduncular pathway and the dopaminergic mesolimbic pathway may jointly mediate the reinforcing properties of addictive drugs. However, the effects of addictive drug on the functioning of the habenulo-interpeduncular pathway have not been well-characterized. Thus, several drugs of abuse (i.e., nicotine, cocaine, amphetamine) have been shown to alter the morphology of the habenulo-interpeduncular pathway, causing selective degeneration of the cholinergic neurons in this area. On the other hand, morphine was shown to alter the neurochemistry of the habenulo-interpeduncular pathway, inducing biphasic changes in acetylcholine release in the interpeduncular nucleus. In order to determine the effects of cocaine, amphetamine and nicotine on cholinergic neurotransmission in the habenulo-interpeduncular pathway, levels of acetylcholine were assessed during microdialysis in freely moving rats. Nicotine (0.1 and 0.4 mg/kg s.c.) produced a dose-dependent decrease in extracellular levels of acetylcholine, while methamphetamine (1 and 4 mg/kg i.p.) produced an increase in acetylcholine release in the interpeduncular nucleus. Cocaine (5 and 20 mg/kg i.p.) produced a biphasic effect on extracellular acetylcholine release, i.e., a low dose enhanced the release of acetylcholine and a high dose decreased its release. These results suggest that the habenulo-intepeduncular pathway may be a common target for drugs of abuse and, by modulating the mesolimbic pathway, may mediate unique aspects of the rewarding effects of different drugs.

Fos immunoreactivity in some locomotor neural centres of 6OHDA-lesioned rats.

In this study, we explore Fos expression (a measure of cell activity) in three nuclei associated with locomotion, namely the zona incerta, pedunculopontine tegmental nucleus and cuneiform nucleus (the latter two form the mesencephalic locomotor region) in hemiparkinsonian rats. Sprague-Dawley rats had small volumes of either saline (control) or 6 hydroxydopamine (6OHDA) injected into the medial forebrain bundle, the major tract carrying dopaminergic nigrostriatal axons. After various post-lesion survival periods, ranging from 2 h to 28 days, rats were perfused with formaldehyde and their brains processed for routine tyrosine hydroxylase and Fos immunocytochemistry. Our results showed a significant increase (P < 0.05) in the number of strongly labelled Fos+ cells in the cuneiform nucleus in the 6OHDA-lesioned cases compared to the controls after 7 and 28 days survival periods. By contrast, there were no significant differences (P > 0.05) in the number of strong-labelled Fos+ cells in the zona incerta and pedunculopontine nucleus of 6OHDA-lesioned rats compared to controls at any survival period. Many of the Fos+ cells within the pedunculopontine and cuneiform nuclei were glutamatergic (35-60%), while none or very few were nitric oxide synthase+. In conclusion, we reveal an increase in the number of strongly labelled Fos+ cells within the cuneiform nucleus of the so-called defensive locomotive system in 6OHDA-lesioned rats. In relation to Parkinson disease, we suggest that this increase is associated with the akinesia or lack of movement seen in patients.

Novel role of brain stem pedunculopontine tegmental adenylyl cyclase in the regulation of spontaneous REM sleep in the freely moving rat.

Physiological activation of kainate receptors and GABA(B) receptors within the pedunculopontine tegmentum (PPT) is involved in regulation of rapid-eye-movement (REM) sleep. Because these two types of receptors may also directly and/or indirectly activate the intracellular cyclic adenosine monophosphate (cAMP) signaling pathway, we hypothesized that this signaling pathway may be involved in the PPT to regulate spontaneous REM sleep. To test this hypothesis, four different doses (0.25, 0.50, 0.75, and 1.0 nmol) of a specific adenylyl cyclase (AC) inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), were microinjected bilaterally (100 nl/site) into the PPT, and the effects on REM sleep in freely moving chronically instrumented rats were quantified. By comparing alterations in the patterns of REM sleep after control injections of vehicle or one of the four different doses of SQ22536, the contributions made by each dose of SQ22536 to REM sleep were evaluated. The results demonstrated that the local microinjection of AC inhibitor SQ22536 into the PPT decreased the total amount of REM sleep for 3 h and increased slow-wave sleep (SWS) for 2 h in a dose-dependent manner. This reduction in REM sleep was due to increased latency and decreased frequency of REM sleep episodes. These results provide evidence that inhibition of AC within the PPT can successfully reduce REM sleep. These findings suggest that activation of the cAMP-signaling pathway within the cholinergic cell compartment of the PPT is an intracellular biochemical/molecular step for generating REM sleep in the freely moving rat.

Long-term effects of striatal lesions on c-Fos immunoreactivity in the pedunculopontine nucleus.

The basal ganglia are a group of subcortical nuclei classically thought to be involved in the control of movement, and they have reciprocal connections with the cortex, thalamus and structures in the brainstem. Recent findings suggest that the basal ganglia interact with structures involved in the control of the sleep-waking cycle. The pedunculopontine tegmental nucleus (PPN) maintains a close relationship with the basal ganglia and is intimately involved in the regulation of wakefulness and REM sleep. This study evaluated changes in activity of PPN neurons following striatal kainic acid-induced lesions. Rats were injected in the anterodorsal striatum with either kainic acid or vehicle and allowed to recover for 7 or 30 days. The results showed an increase in the number of c-Fos+ cells in the PPN 30 days but not 7 days after the striatal lesion, when motor hyperactivity was no longer detected. In addition, we found a significant correlation between the ventricular brain ratio, as an indicator of lesion size, and the number of c-Fos+ cells in the PPN. Furthermore, the spatial distribution of cell types suggested that most c-Fos+ cells in the PPN were not cholinergic. These results provide new insights into the functional relationship between the basal ganglia and the PPN and suggest that the striatum, through its indirect influence on the PPN, may contribute to the regulation of wakefulness and cortical activation.

Regulation of rapid eye movement sleep in the freely moving rat: local microinjection of serotonin, norepinephrine, and adenosine into the brainstem.

STUDY OBJECTIVES: Considerable evidence suggests that rapid eye movement (REM) sleep is induced by glutamatergic activation of cholinergic cells within the pedunculopontine tegmentum (PPT). The aim of this study is to test a popular hypothesis that serotonin, norepinephrine, and adenosine act on PPT cells to regulate REM sleep. This study also tests an alternate hypothesis that serotonin may inhibit REM sleep signs by direct action on the individual REM sleep sign generators. DESIGN: Serotonin, norepinephrine, and adenosine were locally microinjected into the PPT and serotonin was microinjected into the pontine-wave (P-wave) generator (dorsal part of the locus subcoeruleus nucleus) while quantifying the effects on REM sleep and P-wave activity in freely moving rats. SETTING: N/A. PARTICIPANTS: N/A. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Local microinjections of serotonin, norepinephrine, and adenosine into the PPT did not change REM sleep. Microinjection of serotonin into the P-wave generator suppressed P-wave activity but not REM sleep. CONCLUSIONS: The present findings provide direct evidence that serotonin, norepinephrine, and adenosine-induced REM sleep suppression in the behaving rat are not mediated by the PPT. The results also provide direct evidence, for the first time, that serotonin suppresses P-wave activity by acting directly on the P-wave generator. These results suggest that the serotonin-induced inhibition of REM sleep in the freely moving rat is probably not mediated through the mesopontine cholinergic cell compartment but, rather, through individual REM sleep sign generators.