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.

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.

Mesopontine rostromedial tegmental nucleus neurons projecting to the dorsal raphe and pedunculopontine tegmental nucleus: psychostimulant-elicited Fos expression and collateralization.

The mesopontine rostromedial tegmental nucleus (RMTg) is a GABAergic structure in the ventral midbrain and rostral pons that, when activated, inhibits dopaminergic neurons in the ventral tegmental area and substantia nigra compacta. Additional strong outputs from the RMTg to the pedunculopontine tegmental nucleus pars dissipata, dorsal raphe nucleus, and the pontomedullary gigantocellular reticular formation were identified by anterograde tracing. RMTg neurons projecting to the ventral tegmental area express the immediate early gene Fos upon psychostimulant administration. The present study was undertaken to determine if neurons in the RMTg that project to the additional structures listed above also express Fos upon psychostimulant administration and, if so, whether single neurons in the RMTg project to more than one of these structures. We found that about 50% of RMTg neurons exhibiting retrograde labeling after injections of retrograde tracer in the dorsal raphe or pars dissipata of the pedunculopontine tegmental nucleus express Fos after acute methamphetamine exposure. Also, we observed that a significant number of RMTg neurons project both to the ventral tegmental area and one of these structures. In contrast, methamphetamine-elicited Fos expression was not observed in RMTg neurons labeled with retrograde tracer following injections into the pontomedullary reticular formation. The findings suggest that the RMTg is an integrative modulator of multiple rostrally projecting structures.

Mechanism behind gamma band activity in the pedunculopontine nucleus.

The pedunculopontine nucleus (PPN), part of the reticular activating system, modulates waking and paradoxical sleep. During waking and paradoxical sleep, EEG responses are characterized by low-amplitude, high-frequency oscillatory activity in the beta-gamma band range (~20-80 Hz). We have previously reported that gamma band activity may be intrinsically generated by the membrane electroresponsiveness of PPN neurons, and that the neuronal ensemble generates different patterns of gamma activity in response to specific transmitters. This study attempted to identify the voltage-gated calcium and potassium channels involved in the rising and falling phases of gamma oscillations in PPN neurons. We found that all rat (8-14 day) PPN cell types showed gamma oscillations in the presence of TTX and synaptic blockers when membrane potential was depolarized using current ramps. PPN neurons showed gamma oscillations when voltage-clamped at holding potentials above -30 mV, suggesting that their origin may be spatially located beyond voltage-clamp control. The average frequency for all PPN cell types was 23 ± 1 Hz and this increased under carbachol (47 ± 2 Hz; anova df = 64, t = 12.5, P < 0.001). The N-type calcium channel blocker ω-conotoxin-GVIA partially reduced gamma oscillations, while the P/Q-type blocker ω-agatoxin-IVA abolished them. Both ω-CgTX and ω-Aga blocked voltage-dependent calcium currents, by 56 and 52% respectively. The delayed rectifier-like potassium channel blocker α-dendrotoxin also abolished gamma oscillations. In carbachol-induced PPN population responses, ω-agatoxin-IVA reduced higher, and ω-CgTx mostly lower, frequencies. These results suggest that voltage-dependent P/Q- and, to a lesser extent, N-type calcium channels mediate gamma oscillations in PPN.

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.

The rostromedial tegmental nucleus (RMTg), a GABAergic afferent to midbrain dopamine neurons, encodes aversive stimuli and inhibits motor responses.

Separate studies have implicated the lateral habenula (LHb) or amygdala-related regions in processing aversive stimuli, but their relationships to each other and to appetitive motivational systems are poorly understood. We show that neurons in the recently identified GABAergic rostromedial tegmental nucleus (RMTg), which receive a major LHb input, project heavily to midbrain dopamine neurons, and show phasic activations and/or Fos induction after aversive stimuli (footshocks, shock-predictive cues, food deprivation, or reward omission) and inhibitions after rewards or reward-predictive stimuli. RMTg lesions markedly reduce passive fear behaviors (freezing, open-arm avoidance) dependent on the extended amygdala, periaqueductal gray, or septum, all regions that project directly to the RMTg. In contrast, RMTg lesions spare or enhance active fear responses (treading, escape) in these same paradigms. These findings suggest that aversive inputs from widespread brain regions and stimulus modalities converge onto the RMTg, which opposes reward and motor-activating functions of midbrain dopamine neurons.

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.