Increasing cellular lifespan with a flow system in organotypic culture of the Laterodorsal Tegmentum (LDT).

Organotypic brain culture is an experimental tool widely used in neuroscience studies. One major drawback of this technique is reduced neuronal survival across time, which is likely exacerbated by the loss of blood flow. We have designed a novel, tube flow system, which is easily incorporated into the commonly-used, standard semi-permeable membrane culture methodology which has significantly enhanced neuronal survival in a brain stem nucleus involved in control of motivated and arousal states: the laterodorsal tegmental nucleus (LDT). Our automated system provides nutrients and removes waste in a comparatively aseptic environment, while preserving temperature, and oxygen levels. Using immunohistochemistry and electrophysiology, our system was found superior to standard techniques in preserving tissue quality and survival of LDT cells for up to 2 weeks. In summary, we provide evidence for the first time that the LDT can be preserved in organotypic slice culture, and further, our technical improvements of adding a flow system, which likely enhanced perfusion to the slice, were associated with enhanced neuronal survival. Our perfusion system is expected to facilitate organotypic experiments focused on chronic stimulations and multielectrode recordings in the LDT, as well as enhance neuronal survival in slice cultures originating from other brain regions.

Prenatal nicotine exposure alters postsynaptic AMPA receptors and glutamate neurotransmission within the laterodorsal tegmentum (LDT) of juvenile mice.

Despite dissemination of information regarding the harm on fetal development of smoking while pregnant, the number of pregnancies associated with nicotine exposure appears to have stagnated. Presence of nicotine during neural formulation is associated with a higher susceptibility of drug dependence, suggesting an altered development of neurons in circuits involved in saliency and motivation. The laterodorsal tegmental nucleus (LDT) plays a role in coding stimuli valence via afferents to mesolimbic nuclei. Accordingly, alterations in development of neural mechanisms in the LDT could be involved in vulnerability to drug dependency. Therefore, we examined the effect of prenatal nicotine exposure (PNE) on glutamatergic functioning of LDT neurons in mouse brain slices using whole-cell, patch clamp concurrent with fluorescence-based calcium imaging. PNE was associated with larger amplitudes of AMPA-induced currents, and greater AMPA-mediated rises in intracellular calcium. AMPA/NMDA ratios and the AMPA-current rectification index were lower and higher, respectively, consistent with changes in the functionality of AMPA receptors in the PNE, which was substantiated by a greater inhibition of evoked and spontaneous glutamatergic synaptic events by a selective inhibitor of GluA2-lacking AMPA receptors. Paired pulse ratios showed a decreased probability of glutamate release from presynaptic inputs, and fluorescent imaging indicated a decreased action potential-dependent calcium increase associated with PNE. When taken together, our data suggest that PNE alters LDT glutamatergic functioning, which could alter output to mesolimbic targets. Such an alteration could play a role in altered coding of relevancy of drug stimuli that could enhance risk for development of drug dependency.

Development of resurgent and persistent sodium currents in mesencephalic trigeminal neurons.

Sodium channels play multiple roles in the formation of neural membrane properties in mesencephalic trigeminal (Mes V) neurons and in other neural systems. Mes V neurons exhibit conditional robust high-frequency spike discharges. As previously reported, resurgent and persistent sodium currents (INaR and INaP , respectively) may carry small currents at subthreshold voltages that contribute to generation of spike firing. These currents play an important role in maintaining and allowing high-frequency spike discharge during a burst. In the present study, we investigated the developmental changes in tetrodotoxin-sensitive INaR and INaP underlying high-frequency spike discharges in Mes V neurons. Whole-cell patch-clamp recordings showed that both current densities increased one and a half times from postnatal day (P) 0-6 neurons to P7-14 neurons. Although these neurons do not exhibit subthreshold oscillations or burst discharges with high-frequency firing, INaR and INaP do exist in Mes V neurons at P0-6. When the spike frequency at rheobase was examined in firing Mes V neurons, the developmental change in firing frequency among P7-14 neurons was significant. INaR and INaP density at -40 mV also increased significantly among P7-14 neurons. The change to an increase in excitability in the P7-14 group could result from this quantitative change in INaP. In neurons older than P7 that exhibit repetitive firing, quantitative increases in INaR and INaP density may be major factors that facilitate and promote high-frequency firing as a function of age in Mes V neurons.

Age-related changes in nicotine response of cholinergic and non-cholinergic laterodorsal tegmental neurons: implications for the heightened adolescent susceptibility to nicotine addiction.

The younger an individual starts smoking, the greater the likelihood that addiction to nicotine will develop, suggesting that neurobiological responses vary across age to the addictive component of cigarettes. Cholinergic neurons of the laterodorsal tegmental nucleus (LDT) are importantly involved in the development of addiction, however, the effects of nicotine on LDT neuronal excitability across ontogeny are unknown. Nicotinic effects on LDT cells across different age groups were examined using calcium imaging and whole-cell patch clamping. Within the youngest age group (P7-P15), nicotine induced larger intracellular calcium transients and inward currents. Nicotine induced a greater number of excitatory synaptic currents in the youngest animals, whereas larger amplitude inhibitory synaptic events were induced in cells from the oldest animals (P15-P34). Nicotine increased neuronal firing of cholinergic cells to a greater degree in younger animals, possibly linked to development associated differences found in nicotinic effects on action potential shape and afterhyperpolarization. We conclude that in addition to age-associated alterations of several properties expected to affect resting cell excitability, parameters affecting cell excitability are altered by nicotine differentially across ontogeny. Taken together, our data suggest that nicotine induces a larger excitatory response in cholinergic LDT neurons from the youngest animals, which could result in a greater excitatory output from these cells to target regions involved in development of addiction. Such output would be expected to be promotive of addiction; therefore, ontogenetic differences in nicotine-mediated increases in the excitability of the LDT could contribute to the differential susceptibility to nicotine addiction seen across age.

Delays in auditory-cued step initiation are related to increased volume of white matter hyperintensities in older adults.

An increased volume of white matter hyperintensities (WMH) on MRI has been associated with mobility impairments in older adults. The objective of this preliminary study was to investigate the relationship between the volume of WMH and the delays in auditory-cued step initiation. Eight subjects aged 75-83 years participated. The WMH volume in the corticospinal tracts and anterior thalamic radiations were summed. Subjects performed an auditory-cued stepping task that included two simple reaction time (SRT) trials and three choice reaction time (CRT) trials. SRT trials required subjects to step as quickly as possible with the right foot from a symmetric standing position to a single target position in response to an auditory stimulus. For the CRT trials, subjects stepped as quickly as possible to one of two possible locations, depending on the auditory stimulus. The time from the stimulus onset to the reaction time of the anticipatory postural adjustment (APA(RT)) and liftoff (LO) of the right foot was computed for each stimulus. The mean APA(RT) and LO were greater for the CRT steps compared with the SRT steps to the same location. Increases in WMH were significantly associated with larger APA(RT) and LO during both SRT and CRT for both target locations. These data suggest that increased volume of WMH is associated with greater central processing time during voluntary step initiation, and highlight a possible mechanism that can help to explain how damage to white matter tracts affects mobility in older adults.

[Perinatal development of mammillotegmental connections in rats].

Development of direct axonal connections of the hypothalamic mammillary bodies with ventral and dorsal tegmental nuclei of Gudden was studied on fixed rat brains from day 14 of embryonic development until day 10 of postnatal development using the method of diffusion of the lipophilic fluorescent carbocyanine tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The tracer was inserted into the mammillary bodies or into the tegmentum and after incubation in a fixative fluorescent nerve cells and nerve fibers were visualized in the brain tissue. The mammillotegmental tract was found to start developing earlier than other conducting systems of the mammillary bodies. On days 14-15 of embryonic development, it was visualized as a bundle of axons running from the mammillary bodies caudally to the midbrain. A group of neurons in the midbrain tegmentum and their axons going to the mammillary bodies via the mammillary peduncle were first visualized on day 19 of embryonic development. The mammillotegmental tract and mammillary peduncle developed progressively from the moment of birth. Ventral and dorsal tegmental nuclei were formed in the midbrain by day 10 of the postnatal development. Thus, the formation of reciprocal connections of the mammillary bodies with midbrain tegmental nuclei was first described during perinatal development in rats.

Postnatal development of cholinergic neurons in the mesopontine tegmentum revealed by histochemistry.

Cholinergic neurons in the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) play a role in the regulation of several kinds of behavior. Some of them, such as locomotion, motor inhibition or sleep, show dramatic changes at a certain period of postnatal development. To understand the neural substrate for the development of these physiological functions, we studied the development of cholinergic neurons in the LDT and PPT of postnatal and adult rats using histochemical staining of NADPH-diaphorase (NADPH-d) and immunohistochemical staining of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). At postnatal day 1 (P1), ChAT- and VAChT-stained cells localized more dorsally than those of NADPH-d-stained cells, and at P7 their distributions became similar to those of NADPH-d-stained cells. The number of NADPH-d-stained cells increased rapidly after birth, reaching the adult level by P7. In contrast, the number of ChAT- and VAChT-stained cells and the intensity of their staining decreased from P1 to P3 and then increased through P21. The volume of the LDT increased during the second postnatal week. These findings indicate that cholinergic neurons in the LDT develop their cholinergic properties during the second postnatal week and mature functionally thereafter. We discuss these results in light of the several physiological functions regulated by the cholinergic neurons in the mesopontine tegmentum.

Olivary projection from the rostral part of the nucleus of Darkschewitsch in the postnatal rat as revealed through the use of a carbocyanine dye.

Using a carbocyanine dye in postnatal rats, we have shown that the rostral part of the nucleus of Darkschewitsch (ND), consisting of a subnucleus of the so-called "area parafascicularlis prerubralis " and excluded from the rat's ND proper, projects ipsilaterally to the rostral part of the medial accessory olive. The present study suggests the existence of a precise topographic organization from subnuclei of the area parafascicularlis prerublaris to subnuclei of the inferior olive.

Developmental changes in pedunculopontine nucleus (PPN) neurons.

The developmental decrease in rapid-eye-movement (REM) sleep in man occurs between birth and after puberty. We hypothesize that if this decrease in REM sleep does not occur, lifelong increases in REM sleep drive may ensue. Such disorders are characterized by hypervigilance and sensory-gating deficits, such as are present in postpubertal onset disorders like schizophrenia, panic attacks (a form of anxiety disorder), and depression. The decrease in REM sleep in the rat occurs between 10 and 30 days of age. We studied changes in size and physiological properties of pedunculopontine nucleus (PPN) cells involved in the control of arousal, i.e., waking and REM sleep. During the largest decrease in REM sleep (12-21 days), cholinergic PPN neurons doubled in cell area, the hypertrophy peaking at 15-16 days, then decreasing in area by 20-21 days. Noncholinergic PPN cells did not change in area during this period. We confirmed the presence of two populations of PPN neurons based on action potential (AP) duration, with the proportion of short-AP-duration cells increasing and long AP duration decreasing between 12 and 21 days. Most cholinergic and noncholinergic cells had short AP durations. Afterhyperpolarization (AHP) duration became segregated into long and short AHP duration after 15 days. Cells with short AP duration also had short AHP duration. The proportion of PPN cells with Ih current increased gradually, peaking at 15 days, then decreased by 21 days. These changes in morphological and physiological properties are discussed in relation to the developmental decrease in REM sleep.

Ontogeny and distribution of gonadotropin-releasing hormone (GnRH) neuronal systems in the brain of the cichlid fish Cichlasoma dimerus.

Using immunocytochemistry we have described the distribution and ontogeny of three distinct gonadotropin-releasing hormone (GnRH) neural systems, emphasizing the analysis during the period of sex differentiation in the South American cichlid fish Cichlasoma dimerus. In the forebrain a group of neurones immunoreactive to salmon GnRH that formed clusters in the nucleus olfacto retinalis (NOR), was located at the junction of the olfactory bulb and the telencephalon. These neurones differentiated 3 days after fertilization from the olfactory placodes. GnRH immunoreactive neurones along the olfactory nerves through the rostrobasal olfactory bulb were observed on day 4 and at the NOR on day 10. A group of neurones immunoreactive to chicken GnRH II was seen in the dorsal midbrain tegmentum. They originate from the ventricular ependyma between days 5 and 6. These neurones remained close to blood vessels throughout development. Between days 22 and 30 a group of neurones immunoreactive to seabream GnRH was detected in the anterior basal preoptic area. GnRH innervation of the pituitary was detected after the differentiation of preoptic neurones and in coincidence with gonadal differentiation. We hypothesize that the GnRH neural systems have three distinct embryonic origins. Furthermore, we show that the NOR and the midbrain GnRH neurones might have functions other than gonadal development, whereas the preoptic GnRH neurones in C. dimerus might be associated with gonadal sex differentiation.

Discussant--pathophysiologies of Rett syndrome.

Studies on sleep parameters of Rett syndrome revealed hypoactivity of the noradrenaline (NA) and the serotonin (5HT) neuron in early infancy while preserving the function of the dopamine (DA) and the cholinergic neurons of the pons normally. The sleep-wake cycle remains in its development at the level of 4 months of age. Polysomnographies also showed a decrease of the function of the nigrostriatal (NS)-DA neuron in early childhood and suggested the development of receptor supersensitivity in late childhood. Neurohistochemical and neuroimaging (PET) studies revealed the hypofunction of the NS-DA neuron with receptor supersensitivity and of involvement of the cholinergic neurons to the cortical pathology, whereas no substantial pathological or histochemical abnormalities were observed in the NA and the 5HT neurons in the brainstem. The decrease of tyrosine hydroxylase without neurodegenerative changes observed in the substantia nigra of Rett syndrome had similarity to the pathology caused by excitotoxic lesion of the pedunculopontine nuclei (PPN) observed in an animal experiments. Clinically the grade of disability of locomotion was shown to correlate to the grade of the disabilities of language. These clinical manifestations were also correlated to the specific loci of the mutation in the methyl binding domain of the MECP2 gene. In rodents the axons of the brainstem 5HT neuron involved in the morphogenesis of the brain in the early developmental course disappear in neonates without apoptotic or degenerative changes in the neurons. This period corresponds to the first 1.5-2 years in humans. Thus, in Rett syndrome, the primary lesion appears in the brainstem NA and 5HT neurons which affects development of synaptogenesis of the cortex and also dysfunction of the PPN. The latter causes dysfunction of the DA neuron and the cholinergic neuron in the midbrain. The mutation of the MECP2 gene may cause early transcription of the genes which prune the axons of the aminergic neurons for the developmental morphogenesis of the central nervous system in early infancy.

Expression of R-cadherin and N-cadherin by cell groups and fiber tracts in the developing mouse forebrain: relation to the formation of functional circuits.

The expression of R-cadherin and N-cadherin was mapped in the postnatal forebrain of the mouse by immunohistochemistry and in situ hybridization. Results show that the two molecules are expressed in specific and restricted patterns in numerous brain nuclei, gray matter areas and cortical layers that are widely distributed throughout the mouse forebrain at postnatal day 1. The expression pattern of R-cadherin is clearly distinct from that of N-cadherin, but overlap is observed in many areas. In many cortical areas, the two cadherins have a laminar-specific distribution that varies from region to region. In addition, immunohistochemical data revealed expression of R-cadherin protein and N-cadherin protein in the neuropil of many brain regions as well as in the axons that travel in fiber tracts such as the olfactory tract, the anterior commissure, the corpus callosum, the stria terminalis and the fornix. Often, subsets of axons within the same fiber tract differentially express R-cadherin and N-cadherin, with partial overlap of expression. The targets of the cadherin-immunoreactive fiber bundles often contain neuropil as well as cell bodies of neurons that also express the same type(s) of cadherin, suggesting that R-cadherin and N-cadherin may be involved in target recognition and the establishment of connections. Specifically, the expression of R-cadherin and N-cadherin is related to the maturation of thalamocortical sensory pathways, corticofugal pathways, and pathways associated with the hippocampal complex, the piriform cortex, and the amygdala. It is also related to the development of the cell groups associated with these pathways.Together, the results from the present study indicate the possibility that the selective adhesion of neural structures that express the same type(s) of cadherin contributes to the formation of gray matter areas, neural circuits and functional connections in the postnatal forebrain of the mouse.

Perineuronal nets show intrinsic patterns of extracellular matrix differentiation in organotypic slice cultures.

Perineuronal nets (PNs), consisting of extracellular matrix proteoglycans, complexed with hyaluronan and colocalized with tenascins, are associated with distinct neuronal populations in mature mammalian brain. PNs have been shown to appear postnatally during the period of synaptic refinement and myelination, indicating the commencement of mature physiological properties of neurons. Here we show that the developmental patterns of formation of PNs are well preserved in organotypic slice cultures prepared from rats on postnatal day 3-5 and maintained in vitro for 3-10 weeks. Staining of cultures with Wisteria floribunda agglutinin and immunocytochemical detection of chondroitin sulfate proteoglycans revealed developing PNs in the basal forebrain, mesencephalic regions, and the cerebellum after 2 weeks in vitro, and later in the neocortical areas and hippocampus. In contrast, neurons known to be devoid of PNs in the adult rat brain such as cholinergic basal forebrain neurons and catecholaminergic tegmental neurons differentiate without any formation of PNs in slice cultures. We show further that environmental factors influence the development of PNs around the net-associated types of neurons. Notably, chronic depolarization of the cultures, imposed by an elevated concentration of external potassium ions, enhanced the development of PNs. Blocking of calcium channels with magnesium chloride or with the L-type calcium channel blocker nifedipine, suppressed the development of PNs, while a block of voltage-gated sodium channels by tetrodotoxin had no obvious effects. The results show that extracellular matrix components specifically contribute to the organotypic patterns that develop in brain slice cultures. Evidence is provided that the differentiation of PNs is regulated by calcium-dependent signaling.

Extinction of behavior in infant rats: development of functional coupling between septal, hippocampal, and ventral tegmental regions.

Learning of a behavior at a particular age during the postnatal period presumably occurs when the functional brain circuit mediating the behavior matures. The inability to express a learned behavior, such as inhibition, may be accounted for by the functional dissociation of brain regions comprising the circuit. In this study we tested this hypothesis by measuring brain metabolic activity, as revealed by fluorodeoxyglucose (FDG) autoradiography, during behavioral extinction in 12- and 17-d-old rat pups. Subjects were first trained on a straight alley runway task known as patterned single alternation (PSA), wherein reward and nonreward trials alternate successively. They were then injected with FDG and given 50 trials of continuous nonreward (i.e., extinction). Pups at postnatal day 12 (P12) demonstrated significantly slower extinction rates compared to their P17 counterparts, despite the fact that both reliably demonstrated the PSA effect, i.e., both age groups distinguished between reward and nonreward trials during acquisition. Covariance analysis revealed that the dentate gyrus, hippocampal fields CA1-3, subiculum, and lateral septal area were significantly correlated in P17 but not P12 pups. Significant correlations were also found between the lateral septal area, ventral tegmental area, and the medial septal nucleus in P17 pups. Similar correlative patterns were not found in P12 and P17 handled control animals. Taken together, these results suggest that septal, hippocampal, and mesencephalic regions may be functionally dissociated at P12, and the subsequent maturation of functional connectivity between these regions allows for the more rapid expression of behavioral inhibition during extinction at P17.

Calcium-binding proteins in the substantia nigra and ventral tegmental area during development: correlation with dopaminergic compartmentalization.

The importance of calcium in neuronal function has been amply demonstrated in recent years. The discovery of a class of proteins within neurons which bind calcium, therefore, has proven to be a catalyst for the generation of theories and hypotheses regarding mechanisms of neurotoxicity in the CNS. In addition, the distribution of certain calcium-binding proteins changes during neural development, suggesting that they may play a role in organization or pattern generation. We have examined the ontogeny of three related calcium-binding proteins, calbindin-D28, parvalbumin and calretinin, with respect to the ventral and dorsal compartments or tiers of the dopaminergic population in the ventral midbrain. Single and dual-label immunocytochemistry was employed to map the distributions of calcium-binding proteins and tyrosine hydroxylase from E18 through adulthood. The results show that each of the three proteins exhibits a unique developmental sequence and compartment preference, with calbindin D28 clearly related to the later-developing dorsal tier, and parvalbumin and calretinin to the ventral tier of the dopaminergic ventral mesencephalon.

Differential binding profile and internalization process of neurotensin via neuronal and glial receptors.

Two G-protein-coupled receptors for the tridecapeptide neurotensin (NT) have been identified and cloned in mammalian brain: a high-affinity (Kd = 0.3 nM) receptor, sensitive to the antagonist SR 48692 but insensitive to levocabastine, and a lower-affinity (Kd = 2-4 nM) receptor, sensitive to levocabastine but with poor affinity for SR 48692. Although there is good evidence that the high-affinity site is predominantly expressed in neurons, little is known of the cellular localization of the low-affinity receptor. In the present study, we identify by confocal microscopy selective levocabastine-sensitive, SR 48692-resistant binding of a fluorescent derivative of NT (fluo-NT) to a subpopulation of glial fibrillary acidic protein-immunoreactive glial cells grown in culture from the midbrain and cerebral cortex of embryonic and neonatal rats, respectively. We also demonstrate, by combining fluo-NT detection with tyrosine hydroxylase immunofluorescence, that these glial binding sites are differentially regulated from the SR 48692-sensitive NT receptor expressed in the same cultures by mesencephalic dopamine neurons. Whereas the latter undergoes rapid ligand-induced internalization followed by centripetal mobilization of ligand-receptor complexes from processes to perikarya and from perikaryal periphery to cell center, the former induces the formation of cell-surface clusters that fail to internalize. It is concluded that NT may exert its effects on both neurons and astrocytes in the CNS. Whereas NT neural signaling is exerted through high-affinity receptors and may be partly effected through internalization of receptor-ligand complexes, glial signaling is exerted through low-affinity NT receptors and appears to be transduced exclusively at the level of the plasma membrane.

An immunocytochemical study of a G-protein-gated inward rectifier K+ channel (GIRK2) in the weaver mouse mesencephalon.

It has been suggested that a mutation in a G-protein-gated inward rectifier K+ channel (GIRK2) is responsible for inducing cell death in the cerebellum of homozygous weaver (wv/wv) mutant mice. These mice also display a progressive, massive loss of mesencephalic dopaminergic neurones. Using an immunocytochemical method, we detected GIRK2-positive cell bodies and fibres in the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA) of control (+/+) mice. Cell counts of both GIRK2- and tyrosine hydroxylase (TH)-positive neurones demonstrated a marked loss of SNC cell bodies, especially in 12-month-old (12M) wv/wv mice. A considerable proportion of GIRK2-positive cell bodies were preserved, however. In addition, no loss of GIRK2-positive neurones was observed in the VTA of 12M wv/wv mice, despite of a significant reduction in TH-positive cell bodies. These results suggest that expression of the mutated channel is not a sufficient condition to induce cell death in the ventral mesencephalon of the wv/wv mice.

Contribution of the ascending cholinergic pathways in the production of ultrasonic vocalization in the rat.

It has been well documented that cholinergic stimulation of the mediobasal forebrain structures induces 20-30 kHz ultrasonic vocalization in adult rats. If the cholinergic system plays a triggering role for ultrasonic vocalization, the question arises as to where the source of the cholinergic fibres, which innervate the mediobasal forebrain and induce vocalization, is located. In the present study, the role of the ascending cholinergic projection from the ponto-mesencephalic cholinergic nuclei to the mediobasal hypothalamic-preoptic region in production of 22 kHz calls was investigated. Cholinergic neurons were stimulated by local injection of L-glutamate and eventual vocalization was recorded by a S200 bat detector and analyzed sonographically. Intracerebral injection of L-glutamate into the laterodorsal tegmental nucleus induced short latency, 20-30 kHz ultrasonic calls. Sound frequency (pitch) and single call duration of the L-glutamate-induced vocalization did not differ from those obtained by cholinergic stimulation of the mediobasal hypothalamic-preoptic region with carbachol. However, L-glutamate stimulation of the laterodorsal tegmental nucleus was ineffective or less effective in 70% of responses, when the terminal fields in the mediobasal hypothalamic-preoptic region were pretreated with scopolamine, a muscarinic antagonist. The results demonstrate that the ascending cholinergic projection from the laterodorsal tegmental nucleus plays a triggering role for 20-20 kHz vocalization in adult rats.

Postnatal development of mesoaccumbens dopamine neurons in the rat: electrophysiological studies.

The postnatal development of antidromically identified mesoaccumbens dopamine (MADA) neurons were examined with single-unit electrophysiological techniques. Rats were anesthetized with chloral hydrate. The physiological characteristics of 1-, 2-, 4- and 5-week-old rat pups were compared to adults (7-9-weeks-old). The basal discharge rate, conduction velocity, antidromic latency and discharge patterns of MADA neurons were not significantly different among the 4- and 5-week-old and adult MADA neurons. MADA neurons from 1- and 2-week-old pups, however, had significantly lower mean basal discharge rates and significantly lower mean conduction velocities than MADA neurons from the older animals (i.e., 4-weeks old, 5-weeks old and adults). 1- and 2-week-old MADA neurons were also found to have significantly longer mean antidromic latencies than MADA neurons from older animals. Significantly fewer 1- and 2-week-old MADA neurons were found to discharge in a bursting pattern when compared to MADA neurons from older animals. These results indicate that during early postnatal development MADA neurons are spontaneously active, but still physiologically immature. The results of the present study are discussed in the context of previous developmental electrophysiological studies of nigrostriatal dopamine neurons.

Early developmental changes in [3H]nicotine binding in the human brainstem.

Little is known about the developmental profile of nicotinic cholinergic receptors in the developing human brain, despite the potential importance of such information in understanding the pathogenesis of neurological abnormalities or increased risk for the sudden infant death syndrome in offspring exposed to nicotine in utero. In this study, we determined the distribution of [3H]nicotine binding in the developing human brainstem by quantitative tissue autoradiography. In midgestational fetuses, [3H]nicotine binding sites were heavily concentrated in tegmental nuclei related to cardiopulmonary integration, arousal, attention, rapid eye movement sleep, and somatic motor control. Over the last half of gestation, [3H]nicotine binding decreased 60-70% in the tegmental nuclei, with a significant difference in binding between midgestation and early infancy. In contrast, there was essentially no change in [3H]nicotine binding in the major cerebellar-relay nuclei (principal inferior olive and griseum pontis) between the same time-points. Tritium quenching by increasing lipid (myelin) content in tissue sections did not account for the decreases in [3H]nicotine binding in tegmental nuclei. Based upon the high levels of [3H]nicotine binding at midgestation, combined with experimental data demonstrating trophic properties for acetylcholine, we postulate that nAChRs a role in the development of the brainstem tegmentum during this period, and that once this role is fulfilled, nicotinic cholinergic binding decreases and remains low thereafter. Alternatively, nicotinic cholinergic receptors may be critical for other developmentally related functions and/or neurotransmission in the brainstem tegmentum at midgestation. The high levels of [3H]nicotine binding in the brainstem tegmentum at midgestation and its rapidly changing profile over late gestation further suggest that mid-to-late gestation is a developmental period during which this region is likely to be most vulnerable to the harmful effects of nicotine in maternal cigarette smoke. The baseline information provided in this study is potentially relevant towards understanding attention deficits and risk for the sudden infant death syndrome in offspring exposed to cigarette smoke in utero.