Dwayne W. Godwin

Synaptic targets of thalamic reticular nucleus terminals in the visual thalamus of the cat.

A major inhibitory input to the dorsal thalamus arises from neurons in the thalamic reticular nucleus (TRN), which use gamma‐aminobutyric acid (GABA) as a neurotransmitter. We examined the synaptic targets of TRN terminals in the visual thalamus, including the A lamina of the dorsal lateral geniculate nucleus (LGN), the medial interlaminar nucleus (MIN), the lateral posterior nucleus (LP), and the pulvinar nucleus (PUL). To identify TRN terminals, we injected biocytin into the visual sector of the TRN to label terminals by anterograde transport. We then used postembedding immunocytochemical staining for GABA to distinguish TRN terminals as biocytin‐labeled GABA‐positive terminals and to distinguish the postsynaptic targets of TRN terminals as GABA‐negative thalamocortical cells or GABA‐positive interneurons. We found that, in all nuclei, the TRN provides GABAergic input primarily to thalamocortical relay cells (93–100%). Most of this input seems targeted to peripheral dendrites outside of glomeruli. The TRN does not appear to be a significant source of GABAergic input to interneurons in the visual thalamus. We also examined the synaptic targets of the overall population of GABAergic axon terminals (F1 profiles) within these same regions of the visual thalamus and found that the TRN contacts cannot account for all F1 profiles. In addition to F1 contacts on the dendrites of thalamocortical cells, which presumably include TRN terminals, another population of F1 profiles, most likely interneuron axons, provides input to GABAergic interneuron dendrites. Our results suggest that the TRN terminals are ideally situated to modulate thalamocortical transmission by controlling the response mode of thalamocortical cells. J. Comp. Neurol. 440:321–341, 2001.

Neurotransmitters Contained in the Subcortical Extraretinal Inputs to the Monkey Lateral Geniculate Nucleus

The lateral geniculate nucleus (LGN) is the thalamic relay of retinal information to cortex. An extensive complement of nonretinal inputs to the LGN combine to modulate the responsiveness of relay cells to their retinal inputs, and thus control the transfer of visual information to cortex. These inputs have been studied in the most detail in the cat. The goal of the present study was to determine whether the neurotransmitters used by nonretinal afferents to the monkey LGN are similar to those identified in the cat. By combining the retrograde transport of tracers injected into the monkey LGN with immunocytochemical labeling for choline acetyl transferase, brain nitric oxide synthase, glutamic acid decarboxylase, tyrosine hydroxylase, or the histochemical nicotinamide adenine dinucleotide phosphate (NADPH)‐diaphorase reaction, we determined that the organization of neurotransmitter inputs to the monkey LGN is strikingly similar to the patterns occurring in the cat. In particular, we found that the monkey LGN receives a significant cholinergic/nitrergic projection from the pedunculopontine tegmentum, γ‐aminobutyric acid (GABA)ergic projections from the thalamic reticular nucleus and pretectum, and a cholinergic projection from the parabigeminal nucleus. The major difference between the innervation of the LGN in the cat and the monkey is the absence of a noradrenergic projection to the monkey LGN. The segregation of the noradrenergic cells and cholinergic cells in the monkey brainstem also differs from the intermingled arrangement found in the cat brainstem. Our findings suggest that studies of basic mechanisms underlying the control of visual information flow through the LGN of the cat may relate directly to similar issues in primates, and ultimately, humans. J. Comp. Neurol. 424:701–717, 2000.

Unique presynaptic and postsynaptic roles of Group II metabotropic glutamate receptors in the modulation of thalamic network activity

The thalamic reticular nucleus (TRN) is a sheet of GABAergic neurons that project to other TRN neurons and to associated thalamocortical relay nuclei. The TRN receives glutamatergic synaptic inputs from cortex as well as reciprocal inputs from the collaterals of thalamocortical neurons. In addition to ionotropic glutamate receptors, metabotropic glutamate receptors (mGluRs) are present in the TRN circuitry. Using whole cell voltage clamp recordings, we pharmacologically characterized unique pre- and postsynaptic functions for Group II mGluRs (mGluR 2 and mGluR 3) within the TRN circuitry in ferrets. mGluR 2 was found on presynaptic cortical axon terminals in the TRN, where it reduced glutamate release, while mGluR 3 acted postsynaptically on TRN cells to increase membrane conductance. Using miniature inhibitory postsynaptic current analysis, we also found that picrotoxin-sensitive intra-TRN GABA-mediated neurotransmission was not affected by administration of a Group II mGluR agonist, indicating that neither mGluR 2 nor 3 acts on presynaptic GABA-containing terminals within the TRN. Because strong corticothalamic activation is implicated in abnormal thalamic rhythms, we used extracellular recordings in the lateral geniculate nucleus to study the effect of Group II mGluR agonists upon these slow oscillations. We induced approximately 3 Hz spike-and-wave discharge activity through corticothalamic stimulation, and found that such activity was reduced in the presence of the Group II mGluR agonist, (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268). These data indicate that Group II mGluR reduce the impact of corticothalamic excitation, and that they may be a useful target in the reduction of absence-like rhythms.

An Acquired Channelopathy Involving Thalamic T-Type Ca2+ Channels after Status Epilepticus

Some epilepsies are linked to inherited traits, but many appear to arise through acquired alterations in neuronal excitability. Status epilepticus (SE) is associated with numerous changes that promote spontaneous recurrent seizures (SRS), and studies have suggested that hippocampal T-type Ca2+ channels underlie increased bursts of activity integral to the generation of these seizures. The thalamus also contributes to epileptogenesis, but no studies have directly assessed channel alterations in the thalamus during SE or subsequent periods of SRS. We therefore investigated longitudinal changes in thalamic T-type channels in a mouse pilocarpine model of epilepsy. T-type channel gene expression was not affected during SE; however CaV3.2 mRNA was significantly upregulated at both 10 d post-SE (seizure-free period) and 31 d post-SE (SRS-period). Overall T-type current density increased during the SRS period, and the steady-state inactivation shifted from a more hyperpolarized membrane potential during the latent stage, to a more depolarized membrane potential during the SRS period. CaV3.2 functional involvement was verified with CaV3.2 inhibitors that reduced the native T-type current in mice 31 d post-SE, but not in controls. Burst discharges of thalamic neurons reflected the changes in whole-cell currents, and we used a computational model to relate changes observed during epileptogenesis to a decreased tendency to burst in the seizure-free period, or an increased tendency to burst during the period of SRS. We conclude that SE produces an acquired channelopathy by inducing long-term alterations in thalamic T-type channels that contribute to characteristic changes in excitability observed during epileptogenesis and SRS.

Differential response dynamics of corticothalamic glutamatergic synapses in the lateral geniculate nucleus and thalamic reticular nucleus.

The corticothalamic feedback pathway provides excitatory synaptic input to both the thalamic reticular nucleus and the lateral geniculate nucleus. We studied excitatory postsynaptic currents elicited from corticothalamic stimulation in the visual sector of the thalamic reticular nucleus and the lateral geniculate nucleus to compare the response of these neurons to stimulation of their common input pathway. Using whole cell patch clamp recordings in ferret thalamic slices, we compared single excitatory postsynaptic current decay kinetics, presynaptic glutamate release dynamics through paired pulse facilitation and responses to corticothalamic train stimulation. We found that single thalamic reticular nucleus excitatory postsynaptic currents were significantly sharper than lateral geniculate nucleus responses. The mean thalamic reticular nucleus excitatory postsynaptic current decay constant (tau) was 4.9+/-0.5 ms, while the mean lateral geniculate nucleus excitatory postsynaptic current tau value was 11.8+/-0.8 ms. Presynaptic release dynamics as measured by responses to paired stimuli were conserved between the thalamic reticular nucleus and lateral geniculate nucleus. However, facilitating responses to train stimulation were markedly different between nuclei. Lateral geniculate nucleus responses showed proportionately larger facilitation (reaching 842.9 +/- 76.4% of excitatory postsynaptic current 1 amplitude) than thalamic reticular nucleus responses (reaching 223.1 +/- 44.0% of excitatory postsynaptic current 1 amplitude). These data indicate that while the corticothalamic pathway produces excitatory postsynaptic currents in both the thalamic reticular nucleus and lateral geniculate nucleus, other factors uniquely affect the functional integration of the inputs in each nucleus.

Diurnal gene expression patterns of T-type calcium channels and their modulation by ethanol

The transient (T-type) calcium channel participates in the generation of normal brain rhythms as well as abnormal rhythms associated with a range of neurological disorders. There are three different isoforms of T-type channels and all are particularly enriched in the thalamus, which is involved in generating many of these rhythms. We report a novel means of T-type channel regulation in the thalamus that involves diurnal regulation of gene expression. Using real time polymerase chain reaction we detected a diurnal pattern of gene expression for all T-type channel transcripts. The peak of gene expression for the CaV3.1 transcript occurred close to the transition from active to inactive (sleep) states, while expression for both CaV3.2 and CaV3.3 peaked near the transition of inactive to active phase. We assessed the effect of chronic consumption of ethanol on these gene expression patterns by examining thalamic tissues of ethanol-consuming cohorts that were housed with the controls, but which received ethanol in the form of a liquid diet. Ethanol consumption resulted in a significant shift of peak gene expression of approximately 5 h for CaV3.2 toward the normally active phase of the mice, as well as increasing the overall gene expression levels by approximately 1.7-fold. Peak gene expression was significantly increased for both CaV3.2 and CaV3.3. Measurements of CaV3.3 protein expression reflected increases in gene expression due to ethanol. Our results illustrate a novel regulatory mechanism for T-type calcium channels that is consistent with their important role in generating thalamocortical sleep rhythms, and suggests that alterations in the pattern of gene expression of these channels could contribute to the disruption of normal sleep by ethanol.

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.

Development of the cholinergic, nitrergic, and GABAergic innervation of the cat dorsal lateral geniculate nucleus

Cholinergic projections from the brainstem have been shown to be important modulators of visual activity in the dorsal lateral geniculate nucleus (dLGN) of the adult, but little is known about the role of these modulatory inputs during development. We examined the postnatal development of the cholinergic innervation of the dLGN by using an monoclonal antibody against choline acetyl transferase (ChAT). We also investigated the development of GABAergic interneurons in the dLGN by using an antibody against glutamic acid decarboxylase (GAD), and the developmental expression of brain nitric oxide synthase (BNOS) by using an antibody against this enzyme. We found that brainstem cells surrounding the brachium conjunctivum express ChAT at birth, although axons in the dLGN do not express ChAT until the end of the first postnatal week. Cholinergic synaptic contacts were observed as early as the second postnatal week. The number of axons stained with the ChAT antibody increased slowly during the subsequent weeks in the dLGN and reached adult levels by the eighth postnatal week. GABAergic interneurons were present at birth and reached their adult soma size by the third postnatal week. GABAergic fibers are dense at birth but change during development from a diffuse pattern to clustered arrangements that can be recognized as distinct rings of GAD staining by P35. Cellular expression of BNOS was seen within all dLGN laminae during development. The BNOS‐stained cells are tentatively identified as interneurons because their soma sizes were similar to those of GAD‐stained cells. Although cellular BNOS staining remained robust in the C1‐3 laminae through adulthood, cellular expression of BNOS in the A laminae declined during the first five postnatal weeks and remains sparse in the adult. As cellular BNOS staining declined, there was a steady increase in BNOS‐stained fibers, which paralleled the increase of ChAT‐stained fibers that are known to colocalize BNOS in the adult. Our results emphasize the continued transformations of intrinsic as well as extrinsic innervation patterns that occur during the development of the dLGN. J. Comp. Neurol. 418:65–80, 2000.

Disrupted thalamic T-type Ca2+ channel expression and function during ethanol exposure and withdrawal.

Chronic ethanol exposure produces profound disruptions in both brain rhythms and diurnal behaviors. The thalamus has been identified as a neural pacemaker of both normal and abnormal rhythms with low-threshold, transient (T-type) Ca(2+) channels participating in this activity. We therefore examined T-type channel gene expression and physiology in the thalamus of C57Bl/6 mice during a 4-wk schedule of chronic intermittent ethanol exposures in a vapor chamber. We found that chronic ethanol disrupts the normal daily variations of both thalamic T-type channel mRNA levels and alters thalamic T-type channel gating properties. The changes measured in channel expression and function were associated with an increase in low-threshold bursts of action potentials during acute withdrawal periods. Additionally, the observed molecular and physiological alterations in the channel properties in wild-type mice occurred in parallel with a progressive disruption in the normal daily variations in theta (4-9 Hz) power recorded in the cortical electroencephalogram. Theta rhythms remained disrupted during a subsequent week of withdrawal but were restored with the T-type channel blocker ethosuximide. Our results demonstrate that a key ion channel underlying the generation of thalamic rhythms is altered during chronic ethanol exposure and withdrawal and may be a novel target in the management of abnormal network activity due to chronic alcoholism.

The native T-type calcium current in relay neurons of the primate thalamus

The generation of thalamic bursts depends upon calcium currents that flow through transiently open (T)-type calcium channels. In this study, we characterized the native T-type calcium current underlying thalamic burst responses in the macaque monkey. Current clamp recordings from lateral geniculate nucleus (LGN) slices showed characteristic burst responses when relay cells were depolarized from relatively hyperpolarized membrane potentials. These bursts could also be elicited by stimulation of excitatory synaptic inputs to LGN cells. Under voltage clamp conditions, the inactivation kinetics of native currents recorded from primate LGN neurons showed consistency with T-type currents recorded in other mammals and in expression systems. Real-time reverse transcriptase PCR performed on RNA isolated from the LGN (including tissues isolated from magnocellular and parvocellular laminae) detected voltage-dependent calcium channel (Ca(v)) 3.1, Ca(v) 3.2, and Ca(v) 3.3 channel transcripts. Ca(v) 3.1 occurred at relatively higher expression than other isoforms, consistent with in situ hybridization studies in rats, indicating that the molecular basis for burst firing in thalamocortical systems is an important conserved property of primate physiology. Since thalamic bursts have been observed during visual processing as well as in a number of CNS disorders, studies of the expression and modulation of these currents at multiple levels are critical for understanding their role in vision and for the discovery of new treatments for disruptions of thalamic rhythms.