Chronic ethanol drinking reduces native T-type calcium current in the thalamus of nonhuman primates.

BACKGROUND: Chronic ethanol use is known to disrupt normal sleep rhythms, but the cellular basis for this disruption is unknown. An important contributor to normal sleep patterns is a low-threshold calcium current mediated by T-type calcium channels. The T-type calcium current underlies burst responses in thalamic nuclei that are important to spindle propagation, and we recently observed that this current is sensitive to acute low doses of ethanol. METHODS: We used a combination of current clamp and voltage clamp recordings in an in vitro brain slice preparation of the dorsal lateral geniculate nucleus (LGN) of macaque monkeys that have chronically self-administered ethanol to determine whether chronic ethanol exposure may affect T-type currents. RESULTS: Current clamp recordings from the LGN of ethanol naive macaques showed characteristic burst responses. However, recordings from the LGN in macaques that self-administered ethanol revealed a significant attenuation of bursts across a range of voltages (n=5). Voltage clamp recordings from control LGN neurons (n=16) and neurons (n=29) from brain slices from chronically drinking macaques showed no significant differences (P>0.05) in T-type current kinetics or in the membrane resistance of the thalamic cells between the two cohorts. However, mean T-type current amplitude measured in the chronically drinking animals was reduced by 31% (P<0.01). CONCLUSIONS: We conclude that chronic ethanol self-administration reduces calcium currents in thalamic relay cells without altering underlying current kinetics, which may provide a mechanistic framework for the well-documented disruptions in sleep/wake behavior in subjects with chronic ethanol exposure.

Ultrastructural analysis of projections to the pulvinar nucleus of the cat. I: Middle suprasylvian gyrus (areas 5 and 7).

The mammalian pulvinar nucleus (PUL) establishes heavy interconnections with the parietal lobe, but the precise nature of these connections is only partially understood. To examine the distribution of corticopulvinar cells in the cat, we injected the PUL with retrograde tracers. Corticopulvinar cells were located in layers V and VI of a wide variety of cortical areas, with a major concentration of cells in area 7. To examine the morphology and distribution of corticopulvinar terminals, we injected cortical areas 5 or 7 with anterograde tracers. The majority of corticopulvinar axons were thin fibers (type I) with numerous diffuse small boutons. Thicker (type II) axons with fewer, larger boutons were also present. Boutons of type II axons formed clusters within restricted regions of the PUL. We examined corticopulvinar terminals labeled from area 7 at the ultrastructural level in tissue stained for gamma-aminobutyric acid (GABA). By correlating the size of the presynaptic and postsynaptic profiles, we were able to quantitatively divide the labeled terminals into two categories: small and large (RS and RL, respectively). The RS terminals predominantly innervated small-caliber non-GABAergic (thalamocortical cell) dendrites, whereas the RL terminals established complex synaptic arrangements with dendrites of both GABAergic interneurons and non-GABAergic cells. Interpretation of these results using Sherman and Guillery's recent theories of thalamic organization (Sherman and Guillery [1998] Proc Natl Acad Sci U S A 95:7121-7126) suggests that area 7 may both drive and modulate PUL activity.

Thalamocortical cells in the cat pulvinar nucleus transiently express nitric oxide synthase during development.

We examined the postnatal expression of the neuronal form of nitric oxide synthase (nNOS) within the pulvinar and lateral posterior (LP) nuclei of the cat thalamus using immunocytochemical techniques. During the first postnatal month, nNOS was expressed in many cells within the pulvinar nucleus and medial subdivision of the LP nucleus; fewer neurons in the lateral LP nucleus were stained by the nNOS antibody. We examined the pulvinar nucleus to determine what cell types express nNOS. A comparison of the soma sizes of nNOS-stained cells to the overall population of Nissl-stained cells and interneurons (stained with an antibody against glutamic acid decarboxylase) suggests that within the pulvinar nucleus, thalamocortical cells express nNOS during development. In addition, the nNOS antibody stained axon bundles that traverse the pulvinar nucleus to enter the optic radiations, suggesting that thalamocortical cell axons also contain nNOS during development. However, this staining pattern was dramatically reduced by postnatal day 42 and later ages; the size of the remaining nNOS-stained cells was closer to that of interneurons, a subset of which contain nNOS in the adult pulvinar nucleus. This contrasts with our previous findings that nNOS is specifically expressed within interneurons in the developing dorsal lateral geniculate nucleus (LGN) and serves as further confirmation that the pulvinar nucleus and LGN represent distinct categories of thalamic nuclei.

Ethanol influences on native T-type calcium current in thalamic sleep circuitry.

Ethanol is known to disrupt normal sleep rhythms; however, the cellular basis for this influence is unknown. This study uses an in vitro slice preparation coupled with electrophysiological recordings to probe neuronal responses to acute ethanol exposure. Recordings were conducted in ferret and rat thalamic slices, since thalamic circuitry is an integral component of sleep/wake cycles and sleep spindles. A key mediator of spindle wave activity is the low-threshold calcium current (T-type current). The T-type current underlies burst responses in the lateral geniculate and thalamic reticular nuclei that are important in spindle propagation. Whole cell patch recordings in thalamic brain slices revealed that ethanol has a differential, dose-dependent effect on the native T-type current in thalamic relay cells. Low concentrations of ethanol (2.5, 5, and 10 mM) enhance T-type current (n = 35), whereas higher concentrations of ethanol (20 and 50 mM) decrease T-type current (n = 27). To address whether this dose-dependent effect was due to variation between cells, in a subset we verified the differential effect within the same cell (n = 7). In an effort to examine whether the biphasic effects on the current were due to the order of ethanol exposures, we varied the order of high and low ethanol concentrations within the same cell. The ability of ethanol to perturb calcium currents in thalamic relay cells may provide a mechanistic framework for the well documented disruptions in sleep/wake behavior in subjects with ethanol exposure.

Ultrastructure and synaptic targets of tectothalamic terminals in the cat lateral posterior nucleus.

The recent appreciation of the fact that the pulvinar and lateral posterior (LP) nuclei receive two distinct types of cortical input has sparked renewed interest in this region of the thalamus. A key question is whether the primary or "driving" inputs to the pulvinar/LP complex originate in cortical or subcortical areas. To begin to address this issue, we examined the synaptic targets of tectothalamic terminals within the LP nucleus. Tectothalamic terminals were labeled using the anterograde transport of biotinylated dextran amine (BDA) or Phaselous leucoagglutinin placed in the superior colliculus or using immunocytochemical staining for substance P, a neurotransmitter found to be used by the tectothalamic pathway (Hutsler and Chalupa [ 1991] J. Comp. Neurol. 312:379-390). Our results suggest that most tectothalamic terminals are large and occupy a proximal position on the dendritic arbor of LP relay cells. In the medial LP, tectothalamic terminals labeled by the transport of neuronal tracers or substance P immunocytochemistry can form tubular clusters that surround the proximal dendrites of relay cells. In a rostral and lateral subdivision of the lateral LP nucleus (LPl-2), tectothalamic terminals form more typical glomerular arrangements. When compared with existing physiological data, these results suggest that a unique integration of tectal and cortical inputs may contribute to the response properties of LP neurons.

Brain nitric oxide synthase expression in the developing ferret lateral geniculate nucleus: analysis of time course, localization, and synaptic contacts.

Nitric oxide (NO) is a diffusible neurotransmitter that has been implicated in key developmental events, including the refinement of retinogeniculate axons into ON/OFF sublayers in the ferret lateral geniculate nucleus (LGN), and in the formation of eye-specific laminae in other species. To understand the role of NO in the LGN, it is critical to fully characterize the pattern of brain nitric oxide synthase (bNOS) expression within the nucleus, including the phenotype of the neural elements that express it. We have examined the temporal and spatial pattern of bNOS expression in the ferret LGN during the first 6 weeks of postnatal development, and in the adult, by detecting bNOS with a monoclonal antibody as well as beta-nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. We have found that bNOS is expressed in neurons in the A laminae of the LGN as early as postnatal day 7 (P7), a time coincident with eye-specific segregation of retinal axons. This expression continues through P35, with peak somatodendritic expression at P21. Fluorescent double labeling using antibodies to bNOS and glutamic acid decarboxylase indicate that bNOS is expressed in gamma-aminobutyric acid-ergic interneurons within the A laminae. Electron microscopic examination of bNOS-labeled cells showed synaptic contacts from terminals with two distinct morphologic profiles. Expression of bNOS within interneurons that receive contacts from multiple sources indicates that the synaptic circuitry associated with bNOS activation and the potential targets of NO may be more complex than originally thought and supports a potential new role for interneurons as cellular intermediaries in the refinement of pathways in the LGN. Our findings broaden the window of time that bNOS may be active within the developing LGN, suggesting an expanded role for NO during early postnatal development.