An augmented dopamine system function is present prior to puberty in the methylazoxymethanol acetate rodent model of schizophrenia.

Schizophrenia is a disease typically associated with an adolescent onset. Although there have been a considerable number of imaging studies investigating the transition to psychosis in prodromal patients, there are relatively few preclinical studies examining potential mechanisms that may contribute to adolescent onset. We have previously demonstrated, in the methylazoxymethanol acetate (MAM) rodent model of schizophrenia, that an enhanced activity within the ventral hippocampus may underlie the dopamine system hyperfunction, suggested to contribute to positive symptoms in patients. Here we demonstrate that the aberrant regulation of dopamine system function, in MAM-treated rats, is present prior to puberty. Furthermore, we now report that while the afferent regulation of ventral tegmental area dopamine neurons (from the hippocampus and pedunculopontine tegmental area) appears intact in preadolescent rats, the behavioral response to alterations in dopamine system function appears to be attenuated in preadolescent rats. Thus, we posit that the pathological alterations underlying psychosis may be present prior to symptom onset and that the "normal" development of the postsynaptic side of the dopamine system may underlie the transition to psychosis.

Responses of developing pedunculopontine neurons to glutamate receptor agonists.

The pedunculopontine nucleus (PPN) is involved in the generation and maintenance of waking and rapid eye movement (REM) sleep, forming part of the reticular activating system. The PPN receives glutamatergic afferents from other mesopontine nuclei, and glutamatergic input is believed to be involved in the generation of arousal states. We tested the hypothesis that, from postnatal days 9 to 17 in the rat, there are developmental changes in the glutamate receptor subtypes that contribute to the responses of PPN neurons. Whole cell patch-clamp recordings were conducted using brainstem slices from 9- to 17-day-old rats. All cells (types I, II, and III; randomly selected or thalamic-projecting) responded to bath application of the glutamate receptor agonists N-methyl-d-aspartic acid (NMDA) and kainic acid (KA). A developmental decrease in the contribution of the NMDA receptor and developmental increase in the contribution of the KA receptor was observed following electrical stimulation-induced glutamate input. These changes were also observed following bath application in different cell types (randomly selected vs. thalamic-projecting). KA bath application produced an increase in the paired-pulse ratio (PPR) and a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs), suggesting that presynaptic KA autoreceptors may decrease the probability of synaptic glutamate input. In contrast, NMDA application produced no changes in the PPR or mEPSCs. Changes in glutamatergic excitability of PPN cell types could underlie the developmental decrease in REM sleep.

GABAergic modulation of developing pedunculopontine nucleus.

Rapid eye movement sleep decreases dramatically during development. We tested the hypothesis that some of this decrease may be due to GABAergic inhibition of reticular activating system neurons. Recordings of pedunculopontine neurons in vitro showed that the gamma-amino-butyric acid, receptor agonist muscimol depolarized noncholinergic cells early in the developmental decrease in rapid eye movement sleep, and hyperpolarized them later. Most cholinergic cells were hyperpolarized throughout the period tested. The gamma-amino-butyric acid b receptor agonist baclofen hyperpolarized both cholinergic and noncholinergic cells, although the degree of polarization decreased with age. Part of the gradual decrement in rapid eye movement sleep during development may be due in part to the increasing inhibition mediated by gamma-amino-butyric acid, a receptor on pedunculopontine neurons. This influence, however, appears to be mainly on noncholinergic cells.

Muscarinic and nicotinic responses in the developing pedunculopontine nucleus (PPN).

The pedunculopontine nucleus (PPN), the cholinergic arm of the reticular activating system (RAS), is known to modulate waking and rapid eye movement (REM) sleep. REM sleep decreases between 10 and 30 days postnatally in the rat, with the majority occurring between 12 and 21 days. We investigated the possibility that changes in the cholinergic, muscarinic and/or nicotinic, input to PPN neurons could explain at least part of the developmental decrease in REM sleep. We recorded intracellularly from PPN neurons in 12-21 day rat brainstem slices maintained in artificial cerebrospinal fluid (aCSF) and found that application of the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) depolarized PPN neurons early in development, then hyperpolarized PPN neurons by day 21. Most of the effects of DMPP persisted following application of the sodium channel blocker tetrodotoxin (TTX), and in the presence of glutamatergic, serotonergic, noradrenergic and GABAergic antagonists, but were blocked by the nicotinic antagonist mecamylamine (MEC). The mixed muscarinic agonist carbachol (CAR) hyperpolarized all type II (A current) PPN cells and depolarized all type I (low threshold spike-LTS current) and type III (A+LTS current) PPN cells, but did not change effects during the period known for the developmental decrease in REM sleep. The effects of CAR persisted in the presence of TTX but were mostly blocked by the muscarinic antagonist atropine (ATR), and the remainder by MEC. We conclude that, while the nicotinic inputs to the PPN may help modulate the developmental decrease in REM sleep, the muscarinic inputs appear to modulate different types of cells differentially.

Alpha-2 adrenergic regulation of pedunculopontine nucleus neurons during development.

Rapid eye movement sleep decreases between 10 and 30 days postnatally in the rat. The pedunculopontine nucleus is known to modulate waking and rapid eye movement sleep, and pedunculopontine nucleus neurons are thought to be hyperpolarized by noradrenergic input from the locus coeruleus. The goal of the study was to investigate the possibility that a change in alpha-2 adrenergic inhibition of pedunculopontine nucleus cells during this period could explain at least part of the developmental decrease in rapid eye movement sleep. We, therefore, recorded intracellularly in 12-21 day rat brainstem slices maintained in oxygenated artificial cerebrospinal fluid. Putative cholinergic vs. non-cholinergic pedunculopontine nucleus neurons were identified using nicotinamide adenine dinucleotide phosphate diaphorase histochemistry and intracellular injection of neurobiotin (Texas Red immunocytochemistry). Pedunculopontine nucleus neurons also were identified by intrinsic membrane properties, type I (low threshold spike), type II (A) and type III (A+low threshold spike), as previously described. Clonidine (20 microM) hyperpolarized most cholinergic and non-cholinergic pedunculopontine nucleus cells. This hyperpolarization decreased significantly in amplitude (mean+/-S.E.) from -6.8+/-1.0 mV at 12-13 days, to -3.0+/-0.7 mV at 20-21 days. However, much of these early effects (12-15 days) were indirect such that direct effects (tested following sodium channel blockade with tetrodotoxin (0.3 microM)) resulted in hyperpolarization averaging -3.4+/-0.5 mV, similar to that evident at 16-21 days. Non-cholinergic cells were less hyperpolarized than cholinergic cells at 12-13 days (-1.6+/-0.3 mV), but equally hyperpolarized at 20-21 days (-3.3+/-1.3 mV). In those cells tested, hyperpolarization was blocked by yohimbine, an alpha-2 adrenergic receptor antagonist (1.5 microM). These results suggest that the alpha-2 adrenergic receptor on cholinergic pedunculopontine nucleus neurons activated by clonidine may play only a modest role, if any, in the developmental decrease in rapid eye movement sleep. Clonidine blocked or reduced the hyperpolarization-activated inward cation conductance, so that its effects on the firing rate of a specific population of pedunculopontine nucleus neurons could be significant. In conclusion, the alpha-2 adrenergic input to pedunculopontine nucleus neurons appears to consistently modulate the firing rate of cholinergic and non-cholinergic pedunculopontine nucleus neurons, with important effects on the regulation of sleep-wake states.

Prenatal exposure to cigarette smoke affects the physiology of pedunculopontine nucleus (PPN) neurons in development.

Prenatal exposure to cigarette smoke is known to produce lasting arousal, attentional and cognitive deficits in humans. The pedunculopontine nucleus (PPN), as the cholinergic arm of the reticular activating system (RAS), is known to modulate arousal, waking and rapid eye movement (REM) sleep. REM sleep decreases between 10 and 30 days postnatally in the rat, especially at 12-21 days. Pregnant dams were exposed to 350 ml of cigarette smoke for 15 min, 3 times per day, from day E14 until birth, and the pups allowed to mature. Intracellularly recorded PPN neurons in 12-21 day rat brainstem slices were tested for intrinsic membrane properties, including the hyperpolarization-activated cation current Ih, which is known to drive oscillatory activity. Type II (A-current) PPN cells from 12-16 day old offspring of treated animals had a 1/2max Ih amplitude of (mean +/- SE) 4.1 +/- 0.9 mV, while 17-21 day cells had a higher 1/2max Ih of 9.9 +/- 1.1 mV (p < 0.0001). Cells from 12-16 day old control brainstems had a 1/2max Ih of 1.3 +/- 0.1 mV, which was lower (p < 0.05) than in cells from prenatally treated offspring; while 17-21 day old cells from controls had a 1/2max Ih of 3.3 +/- 0.3 mV, which was also lower (p < 0.01) than in cells from prenatally treated offspring. In addition, changes in resting membrane potential [control -65. +/- 0.9 mV (n=32); exposed -55.0 +/- 1.4 mV (n = 27) (p < 0.0001)], and action potential (AP) threshold [control -56.5 +/- 0.7 mV (n = 32), exposed -47.0 +/- 1.4 mV (n = 27) (p < 0.0001)], suggest that prenatal exposure to cigarette smoke induced marked changes in cells in the cholinergic arm of the RAS, rendering them more excitable. Such data could partially explain the differences seen in individuals whose parents smoked during pregnancy, especially in terms of their hypervigilance and increased propensity for attentional deficits and cognitive/behavioral disorders.

Postnatal development of cholinergic afferents from the pedunculopontine tegmental nucleus to the lateralis medialis-suprageniculate nucleus of the feline thalamus.

The lateralis medialis-suprageniculate nuclear complex (LM-Sg) has been shown to receive cholinergic fibers from the pedunculopontine tegmental nucleus (PPT). The majority of terminals of these cholinergic fibers make simple synaptic contact with dendritic profiles, whereas some make contacts with the dendrites of projection neurons and GABAergic interneurons forming a glomerular synaptic complex. In the present study, we investigate the postnatal development of glomerular synaptic complexes in the LM-Sg in association with terminals of the PPT-thalamic projection fibers. We examined the postnatal development of cholinergic innervation as well as GABAergic interneuron innervation in the LM-Sg using antibodies against ChAT and GABA, respectively. Although choline acetyltransferase (ChAT)-positive neurons already exist in the PPT at birth (P0), ChAT-positive fibers in the LM-Sg were observed only after P7. These ChAT-positive fibers gradually increased in number, and almost reached the adult level by postnatal day 28 (P28). GABA-positive interneurons were scattered throughout the LM-Sg at P0, increased in size gradually and reached adult size by P14. Immature glomerulus-like synaptic arrangements appeared at P14. Definite glomeruli, in which ChAT-positive terminals are present, were observed at P28. These results emphasize that interneurons in the LM-Sg grow by P14, and then make neural circuits with cholinergic innervation within the glomerulus by 3-4 weeks.