W. Breckinridge Carden

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. J. Comp. Neurol. 464:472–486, 2003.

Two types of interneurons in the cat visual thalamus are distinguished by morphology, synaptic connections, and nitric oxide synthase content.

The distribution of the neuronal form of the nitric oxide‐synthesizing enzyme, brain nitric oxide synthase (BNOS), was examined in the cat thalamus by using immunocytochemical techniques. BNOS was found in both cells and fibers throughout the visual thalamus. BNOS‐stained cells were found consistently in the C laminae of the lateral geniculate nucleus (LGN), the pulvinar nucleus, and the lateral posterior nucleus (LP). In the A laminae of the LGN, variable numbers of BNOS‐stained cells also could be detected. BNOS‐stained cells were identified as a subset of interneurons because they all stained for glutamic acid decarboxylase (GAD), but not all GAD‐stained cells contained BNOS. The average soma area of BNOS‐stained cells was slightly greater than the average soma area of GAD‐stained cells. BNOS‐stained cells display a distinctive dendritic morphology, which is consistent with previous descriptions of class V neurons (Updyke [1979] J. Comp. Neurol. 186:603–619); they have widespread but fairly sparse arbors of thin, somewhat beaded dendrites. BNOS‐stained cells participate in a distinct synaptic circuitry. Although many GAD‐stained profiles are filled with vesicles and participate in complex synaptic arrangements, known as glomeruli, BNOS‐stained dendrites contain small clusters of vesicles and form dendrodendritic contacts in the extraglomerular neuropil. Thus, there appear to be at least two types of γ‐aminobutyric acidergic interneurons in the visual thalamus of the cat. Interneurons that do not contain BNOS (class III morphology) may exert their effects primarily within synaptic glomeruli (Hamos et al. [1985] Nature 317:618–621), whereas interneurons that contain BNOS (class V morphology) contribute primarily to the extraglomerular neuropil. J. Comp. Neurol 413:83–100, 1999.

LOCATION OF MUSCARINIC TYPE 2 RECEPTORS WITHIN THE SYNAPTIC CIRCUITRY OF THE CAT VISUAL THALAMUS

A cholinergic projection from the parabrachial region (PBR) of the brainstem to the visual thalamus has been studied in great detail during the past 20 years. A number of physiological studies have demonstrated that this projection causes a dramatic change in thalamic activity during the transition from sleep to wakefulness. Additionally, the PBR may mediate more subtle changes in thalamic activity as attentional levels fluctuate during the waking state. The synaptic circuitry underlying these events has been identified in the cat thalamus. However, there is currently no anatomical information regarding the distribution of cholinergic receptors in relation to this circuitry. To begin to understand how the PBR projection modulates thalamic activity, we used immunocytochemical techniques to examine the distribution of muscarinic type 2 (M2) receptors in the visual thalamus of the cat. The distribution of M2 receptors correlates well with previous reports of the distribution of cholinergic terminals in the visual thalamus. At the light microscopic level, dense M2 staining was seen in the neuropil of the dorsal lateral geniculate nucleus (dLGN) and pulvinar nucleus and in somata and proximal dendrites of cells in the thalamic reticular nucleus (TRN). In the dLGN and pulvinar nucleus, we quantitatively analyzed the distribution of M2 receptors using electron microscopy. Postembedding immunocytochemistry for gamma aminobutyric acid (GABA) was used to determine whether M2 receptors are present on interneurons or thalamocortical cells. In particular, we examined the distribution of M2 receptors with respect to the known sites of PBR terminations. The dendrites of both thalamocortical cells and interneurons were stained for the M2 receptors in both the glomerular and extraglomerular neuropil. However, the densest staining was found in glomerular GABAergic profiles that displayed the morphology associated with interneuron dendritic terminals (F2 profiles). Our data suggest that M2 receptors play an important role both in blocking thalamic spindle oscillations and in increasing the efficacy of signal transmission during increased attentional states. J. Comp. Neurol. 410:431–443, 1999.

Y retinal terminals contact interneurons in the cat dorsal lateral geniculate nucleus

One of the largest influences on dorsal lateral geniculate nucleus (dLGN) activity comes from interneurons, which use the neurotransmitter γ‐aminobutyric acid (GABA). It is well established that X retinogeniculate terminals contact interneurons and thalamocortical cells in complex synaptic arrangements known as glomeruli. However, there is little anatomical evidence for the involvement of dLGN interneurons in the Y pathway. To determine whether Y retinogeniculate axons contact interneurons, we injected the superior colliculus (SC) with biotinylated dextran amine (BDA) to backfill retinal axons, which also project to the SC. Within the A lamina of the dLGN, this BDA labeling allowed us to distinguish Y retinogeniculate axons from X retinogeniculate axons, which do not project to the SC. In BDA‐labeled tissue prepared for electron microscopic analysis, we subsequently used postembedding immunocytochemical staining for GABA to distinguish interneurons from thalamocortical cells. We found that the majority of profiles postsynaptic to Y retinal axons were GABA‐negative dendrites of thalamocortical cells (117/200 or 58.5%). The remainder (83/200 or 41.5%) were GABA‐positive dendrites, many of which contained vesicles (59/200 or 29.5%). Thus, Y retinogeniculate axons do contact interneurons. However, these contacts differed from X retinogeniculate axons, in that triadic arrangements were rare. This indicates that the X and Y pathways participate in unique circuitries but that interneurons are involved in the modulation of both pathways. J. Comp. Neurol. 430:85–100, 2001.

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.

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 γ‐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 Guillerys 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. J. Comp. Neurol. 485:87–107, 2005.

A novel means of Y cell identification in the developing lateral geniculate nucleus of the cat

We examined the postnatal development of putative Y cells in the dorsal lateral geniculate nucleus (dLGN) using the SMI-32 antibody, which has been demonstrated in the adult cat to stain cells with Y cell morphology. At birth, SMI-32 stained cells were concentrated in the interlaminar zones. During postnatal development, the SMI-32 staining gradually becomes more disperse and by P21 stained cells are found throughout the A and magnocellular C laminae. By the end of the first postnatal week, and in all later ages examined, the SMI-32 stained cells were significantly larger than the overall population of Nissl stained cells and interneurons (stained with an antibody against glutamic acid decarboxylase). Postnatal SMI-32 staining revealed a dramatic increase in soma sizes and the expansion of putative geniculate Y cell dendritic arbors that continued past the second postnatal month. In contrast, the growth of interneurons appeared to be complete by 3-4 postnatal weeks, at which time cell somas stained with SMI-32 have only reached a little over one half of their adult size. Similar to the adult cat, SMI-32 appears to selectively stain the Y cell population during development and may provide a useful morphological marker to examine the participation of Y cells in the developing postnatal circuitry of the dLGN. This further establishes the cat dLGN as a novel model system to study the normal function and pathological reorganization of neurofilaments.

Synaptic targets of cholinergic terminals in the cat lateral posterior nucleus.

We examined profiles in the neuropil of the lateral division of the lateral posterior (LP) nucleus of the cat stained with antibodies against choline acetyl transferase (ChAT) or γ‐aminobutyric acid (GABA), and several differences in the synaptic circuitry of the lateral LP nucleus compared with the pulvinar nucleus and lateral geniculate nucleus (LGN) were identified. In the lateral LP nucleus, there are fewer glomerular arrangements, fewer GABAergic terminals, and fewer cholinergic terminals. Correspondingly, the neuropil of the lateral LP nucleus appears to be composed of a higher percentage of small type I cortical terminals (RS profiles). Similar to the pulvinar nucleus and the LGN, the cholinergic terminals present in the lateral LP nucleus contact both GABA‐negative profiles (thalamocortical cells; 74%) and GABA‐positive profiles (interneurons; 26%). However, in contrast to the pulvinar nucleus and the LGN, the majority of cholinergic terminals in the lateral LP nucleus contact small‐caliber dendritic shafts outside of glomeruli (60 of 82; 73%). Consequently, most cholinergic terminals are in close proximity to RS profiles. Therefore, whereas the cholinergic input to the LGN and pulvinar nucleus appears to be positioned to selectively influence the response of thalamocortical cells to terminals that innervate glomeruli (retinal terminals or large type II cortical terminals), the cholinergic input to the lateral LP nucleus may function primarily in the modulation of responses to terminals that innervate distal dendrites (small type I cortical terminals). J. Comp. Neurol. 410:31–41, 1999.

The location of muscarinic type 2 receptors within the synaptic circuitry of the cat lateral posterior nucleus.

The ultrastructural distribution of the muscarinic type 2 acetylcholine receptor (M2) was examined in the lateral division of the lateral posterior (LP) nucleus of the cat thalamus, using immunocytochemistry. Postembedding immunocytochemical staining for gamma-aminobutyric acid (GABA) further characterized M2 stained profiles. M2 receptors were predominately found on small caliber (presumably distal) dendritic arbors of thalamocortical cells and interneurons in the lateral LP nucleus. While glomeruli were not abundant in the lateral LP nucleus, occasionally they contained dendritic terminals of interneurons (F2 profiles) stained for M2 receptors. Some GABAergic terminals throughout the neuropil also stained for M2 receptors. The location of M2 receptors correlates well with the cholinergic innervation of the lateral LP nucleus and suggests that muscarinic modulation of visual signals differs in the lateral LP nucleus as compared with the lateral geniculate and pulvinar nuclei.