Impact of prenatal nicotine on the structure of midbrain dopamine regions in the rat.

In utero exposure of rats to nicotine (NIC) provides a useful animal model for studying the impact of smoking during pregnancy on human offspring. Certain sequelae of prenatal NIC exposure suggest an impact on the development of the midbrain dopamine (DA) system, which receives a robust cholinergic innervation from the mesopontine tegmentum. We therefore investigated whether prenatal NIC induced structural changes in cells and synapses within the midbrain that persisted into adulthood. Osmotic minipumps delivering either sodium bitartrate (vehicle; VEH) or NIC bitartrate at 2 mg/kg/day were implanted into nine timed-pregnant dams at E4. At birth, rat pups were culled to litters of six males each, and the litters were cross-fostered. Plasma levels of NIC and cotinine from killed pups provided evidence of NIC exposure in utero. Pups separated from dams at weaning showed a trend toward reduced locomotor activity at this time point but not when tested again in adulthood. Adult rats were killed for anatomical studies. Estimates of brain size and volume did not vary with NIC treatment. Midbrain sections stained for Nissl or by immunoperoxidase for tyrosine hydroxylase and analyzed using unbiased stereology revealed no changes in volume or cell number in the substantia nigra compacta or ventral tegmental area as a result of NIC exposure. Within the ventral tegmental area, electron microscopic physical disector analysis showed no significant differences in the number of axon terminals or the number of asymmetric (putative excitatory) or symmetric (putative inhibitory) synapses. Although too infrequent to estimate by unbiased stereology, no obvious difference in the proportion of cholinergic axons was noted in NIC- versus VEH-treated animals. These data suggest that activation of nicotinic receptors during prenatal development induces no significant modifications in the structure of cells in the ventral midbrain when assessed in adulthood.

The inhibitory influence of the lateral habenula on midbrain dopamine cells: ultrastructural evidence for indirect mediation via the rostromedial mesopontine tegmental nucleus.

The lateral habenula (LHb) provides an important source of negative reinforcement signals to midbrain dopamine (DA) cells in the substantia nigra and ventral tegmental area (VTA). This profound and consistent inhibitory influence involves a disynaptic connection from glutamate neurons in the LHb to some population of γ-aminobutyric acid (GABA) cells that, in turn, innervates DA neurons. Previous studies demonstrated that the GABA cells intrinsic to the VTA receive insufficient synaptic input from the LHb to serve as the primary source of this intermediate connection. In this investigation, we sought ultrastructural evidence supporting the hypothesis that a newly identified region of the brainstem, the rostromedial mesopontine tegmental nucleus (RMTg), is a more likely candidate for inhibiting midbrain DA cells in response to LHb activation. Electron microscopic examination of rat brain sections containing dual immunoreactivity for an anterograde tracing agent and a phenotypic marker revealed that: 1) more than 55% of the synapses formed by LHb axons in the RMTg were onto GABA-labeled dendrites; 2) more than 80% of the synapses formed by RMTg axons in the VTA contacted dendrites immunoreactive for the DA synthetic enzyme tyrosine hydroxylase; and 3) nearly all RMTg axons formed symmetric synapses and contained postembedding immunoreactivity for GABA. These findings indicate that the newly identified RMTg region is an intermediate structure in a disynaptic pathway that connects the LHb to VTA DA neurons. The results have important implications for understanding mental disorders characterized by a dysregulation of reward circuitry involving LHb and DA cell populations.

Periaqueductal gray afferents synapse onto dopamine and GABA neurons in the rat ventral tegmental area.

The midbrain central gray (periaqueductal gray; PAG) mediates defensive behaviors and is implicated in the rewarding effects of opiate drugs. Projections from the PAG to the ventral tegmental area (VTA) suggest that this region might also regulate behaviors involving motivation and cognition. However, studies have not yet examined the morphological features of PAG axons in the VTA or whether they synapse onto dopamine (DA) or GABA neurons. In this study, we injected anterograde tracers into the rat PAG and used immunoperoxidase to visualize the projections to the VTA. Immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA was then used to identify the phenotype of innervated cells. Electron microscopic examination of the VTA revealed axons labeled anterogradely from the PAG, including myelinated and unmyelinated fibers and axon varicosities, some of which formed identifiable synapses. Approximately 55% of these synaptic contacts were of the symmetric (presumably inhibitory) type; the rest were asymmetric (presumably excitatory). These findings are consistent with the presence of both GABA and glutamate projection neurons in the PAG. Some PAG axons contained dense-cored vesicles indicating the presence of neuropeptides in addition to classical neurotransmitters. PAG projections synapsed onto both DA and GABA cells with no obvious selectivity, providing the first anatomical evidence for these direct connections. The results suggest a diverse nature of PAG physiological actions on midbrain neurons. Moreover, as both the VTA and PAG are implicated in the reinforcing actions of opiates, our findings provide a potential substrate for some of the rewarding effects of these drugs.

Lateral habenula projections to dopamine and GABA neurons in the rat ventral tegmental area.

Ventral tegmental area (VTA) dopamine (DA) neurons and their forebrain projections are critically involved in reward processing and cognitive functions. Descending projections from the lateral habenula (LHb) play a central role in inhibiting DA cell activity in response to the absence of expected rewards. As LHb efferents are reportedly glutamatergic, their ability to inhibit DA cells would theoretically require a disynaptic connection involving VTA GABA neurons and their local collateral inputs to DA cells. We therefore used anterograde tract-tracing from the LHb to investigate the relative selectivity of LHb synapses onto GABA versus DA VTA neurons. LHb axons were visualized using immunoperoxidase, and DA and GABA cells were marked by immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA, respectively. By ultrastructural analysis, 16% of LHb axons were observed to form synaptic contacts in the VTA, and most of these were of an intermediate morphological type that did not exhibit definitive asymmetric or symmetric character. LHb axons synaptically targeted TH- and GABA-labeled dendrites to a comparable extent (45 and 52% observed incidence, respectively). Pre-embedding immunogold labeling for the vesicular glutamate transporter type 2 and post-embedding immunogold staining for GABA confirmed that approximately 85% of LHb terminals were glutamatergic and not GABAergic. These results suggest that the robust inhibition of DA cells evoked by the LHb is unlikely to arise from a selective innervation of VTA GABA neurons. Moreover, the LHb may mediate a direct excitation of DA cells that is over-ridden by indirect inhibition originating from an extrinsic source.

Ultrastructural analysis of local collaterals of rat ventral tegmental area neurons: GABA phenotype and synapses onto dopamine and GABA cells.

Local synapses formed by nondopamine cells within the ventral tegmental area (VTA) are thought to provide an important regulatory influence on the activity patterns of dopamine (DA) neurons. However, ultrastructural confirmation of intra-areal synapses formed by VTA neurons is lacking, and the synaptic targets of these connections have not been examined. We performed discrete injections of the specific anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) and used electron microscopy to visualize immunoperoxidase labeling within the local collaterals of VTA cells. The phenotype of target neurons was determined by immunogold-silver labeling for GABA or for tyrosine hydroxylase within DA neurons. Within or immediately adjacent to the VTA injection sites, PHAL was incorporated into the soma and dendrites of both GABA and DA cells. Tracer was also detected within myelinated and unmyelinated axons as well as axon terminals. Some labeled terminals formed identifiable synapses, the majority of which (78%) had symmetric morphology (presumably inhibitory). Both DA and GABA dendrites were contacted by these intrinsic axons. Postembedding immunogold labeling verified that local axon collaterals arose mainly from GABA cells (DA neurons are not known to issue recurrent collaterals). Nevertheless, a few synapses with asymmetric morphology (presumably excitatory) were also noted; whether these derive from local glutamate neurons requires further investigation. Hence, our data provide ultrastructural support for the long standing assumption that GABA VTA neurons synapse locally onto DA cells. The findings also suggest the presence of disinhibitory and possibly excitatory circuitry intrinsic to the VTA.

Cholinergic axons in the rat ventral tegmental area synapse preferentially onto mesoaccumbens dopamine neurons.

Cholinergic afferents to the ventral tegmental area (VTA) contribute substantially to the regulation of motivated behaviors and the rewarding properties of nicotine. These actions are believed to involve connections with dopamine (DA) neurons projecting to the nucleus accumbens (NAc). However, this direct synaptic link has never been investigated, nor is it known whether cholinergic inputs innervate other populations of DA and gamma-aminobutyric acid (GABA) neurons, including those projecting to the prefrontal cortex (PFC). We addressed these questions by using electron microscopic analysis of retrograde tract-tracing and immunocytochemistry for the vesicular acetylcholine transporter (VAChT) and for tyrosine hydroxylase (TH) and GABA. In tissue labeled for TH, VAChT(+) terminals frequently synapsed onto DA mesoaccumbens neurons but only seldom contacted DA mesoprefrontal cells. In tissue labeled for GABA, one-third of VAChT(+) terminals innervated GABA-labeled dendrites, including both mesoaccumbens and mesoprefrontal populations. VAChT(+) synapses onto DA and mesoaccumbens neurons were more commonly of the asymmetric (presumed excitatory) morphological type, whereas VAChT(+) synapses onto GABA cells were more frequently symmetric (presumed inhibitory or modulatory). These findings suggest that cholinergic inputs to the VTA mediate complex synaptic actions, with a major portion of this effect likely to involve an excitatory influence on DA mesoaccumbens neurons. As such, the results suggest that natural and drug rewards operating through cholinergic afferents to the VTA have a direct synaptic link to the mesoaccumbens DA neurons that modulate approach behaviors.

Laterodorsal tegmental projections to identified cell populations in the rat ventral tegmental area.

Projections from the laterodorsal tegmentum (LDT) to the ventral tegmental area (VTA) contribute to the activity of dopamine (DA) and GABA cells and, hence, to the affective and cognitive functions of this region. LDT afferents arise from neurochemically diverse cell types and mediate multiple functional influences. However, the VTA cell populations that receive LDT afferents are unknown and were investigated here by anterograde and retrograde tract-tracing in combination with immunocytochemistry to distinguish DA and GABA cells. Approximately 50% of the LDT to VTA pathway formed asymmetric, presumably excitatory synapses that innervated DA and GABA cells in rough proportion to their representation within the VTA. This portion of the LDT innervation appeared to selectively target DA but not GABA mesoaccumbens neurons and provide a relatively nonselective input to both DA and GABA mesoprefrontal cells. The remaining LDT axons formed symmetric, presumably inhibitory synapses with a different pattern of cellular targets that included a preferential input to GABA neurons of both mesoaccumbens and mesoprefrontal populations and an apparently selective innervation of mesoprefrontal and not mesoaccumbens DA neurons. These data suggest that the LDT mediates a convergent excitatory and inhibitory influence on both mesoprefrontal DA and GABA cells but a divergent impact on mesoaccumbens neurons that is likely to excite DA cells and inhibit GABA neurons. Combined with our previous description of prefrontal cortical afferents, our data also indicate that mesoaccumbens DA neurons receive putative excitatory drive from the LDT, whereas mesoprefrontal DA cells receive convergent excitation from both cortical and brainstem sources.

Anatomical substrates for glutamate-dopamine interactions: evidence for specificity of connections and extrasynaptic actions.

For normal regulation of motor, affective, and cognitive functions, dopamine provides an essential modulation of glutamate transmission within multiple brain regions. This paper will review three principal anatomical substrates for such interactions. First, dopamine modulates the activity of glutamate neurons within the cerebral cortex. Evidence will be reviewed for dopamine regulation of pyramidal neurons in the prefrontal cortex via synaptic and extrasynaptic mechanisms and through indirect effects mediated by GABA cells. Second, glutamate neurons innervate dopamine cells within the ventral tegmental area. Evidence will be described for selective glutamate input from the prefrontal cortex or the brain stem tegmentum to different populations of dopamine cells. The third level of interaction occurs within target regions via convergent synaptic or extrasynaptic regulation of common neurons. Such convergence will be reviewed for the basal ganglia, prefrontal cortex, and amygdala. Together, these substrates for glutamate-dopamine interactions provide several mechanisms for normal regulation of brain function. Sites of modulatory interaction between dopamine and glutamate also suggest circuit alterations that might contribute to the pathophysiology of mental health disorders and provide potential sites for therapeutic intervention in these conditions.

Programming neural Hoxd10: in vivo evidence that early node-associated signals predominate over paraxial mesoderm signals at posterior spinal levels.

Studies of the programming of Hox patterns at anterior spinal levels suggest that these events are accomplished through an integration of Hensen's node-derived and paraxial mesoderm signaling. We have used in vivo tissue manipulation in the avian embryo to examine the respective roles of node- derived and other local signals in the programming of a Hox pattern at posterior spinal levels. Hoxd10 is highly expressed in the lumbosacral (LS) spinal cord and adjacent paraxial mesoderm. At stages of LS neural tube formation (stages 12-14), the tailbud contains the remnants of Hensen's node and the primitive streak. Hoxd10 expression was analyzed after transposition of LS neural segments with and without the tailbud, after isolation of normally positioned LS segments from the stage 13 tailbud, and after axial displacement of posterior paraxial mesoderm. Data suggest that inductive signals from the tailbud are primarily responsible for the programming of Hoxd10 at neural plate and the earliest neural tube stages. After these stages, the LS neural tube appears to differ from more anterior neural segments in its lack of dependence on Hox-inductive signals from local tissues, including paraxial mesoderm. Our data also suggest that a graded system of repressive signals for posterior Hox genes is present at cervical and thoracic levels and likely to originate from paraxial mesoderm.