Martha E. Bickford

Pulvinar projections to the striatum and amygdala in the tree shrew.

Visually guided movement is possible in the absence of conscious visual perception, a phenomenon referred to as “blindsight.” Similarly, fearful images can elicit emotional responses in the absence of their conscious perception. Both capabilities are thought to be mediated by pathways from the retina through the superior colliculus (SC) and pulvinar nucleus. To define potential pathways that underlie behavioral responses to unperceived visual stimuli, we examined the projections from the pulvinar nucleus to the striatum and amygdala in the tree shrew (Tupaia belangeri), a species considered to be a prototypical primate. The tree shrew brain has a large pulvinar nucleus that contains two SC-recipient subdivisions; the dorsal (Pd) and central (Pc) pulvinar both receive topographic (“specific”) projections from SC, and Pd receives an additional non-topographic (“diffuse”) projection from SC (Chomsung et al., 2008). Anterograde and retrograde tract tracing revealed that both Pd and Pc project to the caudate and putamen, and Pd, but not Pc, additionally projects to the lateral amygdala. Using immunocytochemical staining for substance P (SP) and parvalbumin (PV) to reveal the patch/matrix organization of tree shrew striatum, we found that SP-rich/PV-poor patches interlock with a PV-rich/SP-poor matrix. Confocal microscopy revealed that tracer-labeled pulvino-striatal terminals preferentially innervate the matrix. Electron microscopy revealed that the postsynaptic targets of tracer-labeled pulvino-striatal and pulvino-amygdala terminals are spines, demonstrating that the pulvinar nucleus projects to the spiny output cells of the striatum matrix and the lateral amygdala, potentially relaying: (1) topographic visual information from SC to striatum to aid in guiding precise movements, and (2) non-topographic visual information from SC to the amygdala alerting the animal to potentially dangerous visual images.

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.

Synaptic development of the mouse dorsal lateral geniculate nucleus

The dorsal lateral geniculate nucleus (dLGN) of the mouse has emerged as a model system in the study of thalamic circuit development. However, there is still a lack of information regarding how and when various types of retinal and nonretinal synapses develop. We examined the synaptic organization of the developing mouse dLGN in the common pigmented C57/BL6 strain, by recording the synaptic responses evoked by electrical stimulation of optic tract axons, and by investigating the ultrastructure of identified synapses. At early postnatal ages (P14), when optic tract stimulation routinely evoked an excitatory postsynaptic potential/inhibitory postsynaptic potential (EPSP/IPSP) sequence, with the latter having both a GABAA and GABAB component. Electrophysiological and ultrastructural observations were consistent. At P7, many synapses were present, but synaptic profiles lacked the ultrastructural features characteristic of the adult dLGN, and little γ‐aminobutyric acid (GABA) could be detected by using immunocytochemical techniques. In contrast, by P14, GABA staining was robust, mature synaptic profiles of retinal and nonretinal origin were easily distinguished, and the size and proportion of synaptic contacts were similar to those of the adult. The emergence of nonretinal synapses coincides with pruning of retinogeniculate connections, and the transition of retinal activity from spontaneous to visually driven. These results indicate that the synaptic architecture of the mouse dLGN is similar to that of other higher mammals, and thus provides further support for its use as a model system for visual system development. J. Comp. Neurol. 518:622–635, 2010.

Comparison of the ultrastructure of cortical and retinal terminals in the rat dorsal lateral geniculate and lateral posterior nuclei

We compared the ultrastructure and synaptic targets of terminals of cortical or retinal origin in the rat dorsal lateral geniculate nucleus (LGN) and lateral posterior nucleus (LPN). Following injections of biotinylated dextran amine (BDA) into cortical area 17, two types of corticothalamic terminals were labeled by anterograde transport. Type I terminals, found throughout the LGN and LPN, were small, drumstick‐shaped terminals that extended from thin axons. At the ultrastructural level in both the LGN and LPN, labeled type I corticothalamic terminals were observed to be small profiles that contained densely packed round vesicles (RS profiles) and contacted small‐caliber dendrites. In tissue stained for gamma amino butyric acid (GABA) using postembedding immunocytochemical techniques, most dendrites postsynaptic to type I corticothalamic terminals did not contain GABA (97%). Type II corticothalamic terminals, found only in the LPN, were large terminals that sometimes formed clusters. At the ultrastructural level, type II terminals were large profiles that contained round vesicles (RL profiles) and contacted large‐caliber dendrites, most of which did not contain GABA (98%). Retinogeniculate terminals, identified by their distinctive pale mitochondria, were similar to type II corticothalamic terminals except that 26% of their postsynaptic targets were vesicle‐containing profiles that contained GABA (F2 profiles). Our results suggest that type I corticothalamic terminals are very similar across nuclei but that the postsynaptic targets of RL profiles vary. Comparison of the responses to retinal inputs in the LGN and to layer V cortical inputs in the LPN may provide a unique opportunity to determine the function of interneurons in the modulation of retinal signals and, in addition, may provide insight into the signals relayed by cortical layer V. J. Comp. Neurol. 460:394–409, 2003.

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.

Ultrastructural Examination of Diffuse and Specific Tectopulvinar Projections in the Tree Shrew

Two pathways from the superior colliculus (SC) to the tree shrew pulvinar nucleus have been described, one in which the axons terminate in dense (or specific) patches and one in which the axon arbors are more diffusely organized (Luppino et al. [1988] J. Comp. Neurol. 273:67–86). As predicted by Lyon et al. ([2003] J. Comp. Neurol. 467:593–606), we found that anterograde labeling of the diffuse tectopulvinar pathway terminated in the acetylcholinesterase (AChE)‐rich dorsal pulvinar (Pd), whereas the specific pathway terminated in the AChE‐poor central pulvinar (Pc). Injections of retrograde tracers in Pd labeled non‐γ‐aminobutyric acid (GABA)‐ergic wide‐field vertical cells located in the lower stratum griseum superficiale and stratum opticum of the medial SC, whereas injections in Pc labeled similar cells in more lateral regions. At the ultrastructural level, we found that tectopulvinar terminals in both Pd and Pc contact primarily non‐GABAergic dendrites. When present, however, synaptic contacts on GABAergic profiles were observed more frequently in Pc (31% of all contacts) compared with Pd (16%). Terminals stained for the type 2 vesicular glutamate transporter, a potential marker of tectopulvinar terminals, also contacted more GABAergic profiles in Pc (19%) compared with Pd (4%). These results provide strong evidence for the division of the tree shrew pulvinar into two distinct tectorecipient zones. The potential functions of these pathways are discussed. J. Comp. Neurol. 510:24–46, 2008.

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.

Relative distribution of synapses in the pulvinar nucleus of the cat: Implications regarding the “driver/modulator” theory of thalamic function

To provide a quantitative comparison of the synaptic organization of “first‐order” and “higher‐order” thalamic nuclei, we followed bias‐corrected sampling methods identical to a previous study of the cat dorsal lateral geniculate nucleus (dLGN; Van Horn et al. [ 2000 ] J. Comp. Neurol. 416:509–520) to examine the distribution of terminal types within the cat pulvinar nucleus. We observed the following distribution of synaptic contacts: large terminals that contain loosely packed round vesicles (RL profiles), 3.5%; presynaptic profiles that contain densely packed pleomorphic vesicles (F1 profiles), 7.3%; profiles that could be both presynaptic and postsynaptic that contain loosely packed pleomorphic vesicles (F2 profiles), 5.0%; and small terminals that contain densely packed round vesicles (RS profiles), 84.2%. Postembedding immunocytochemistry for γ‐aminobutyric acid (GABA) was used to distinguish the postsynaptic targets as thalamocortical cells or interneurons. The distribution of synaptic contacts on thalamocortical cells was as follows: RL profiles, 2.1%; F1 profiles, 6.9%; F2 profiles, 5.4%; and RS profiles, 85.6%. The distribution of synaptic contacts on interneurons was as follows: RL profiles, 11.8%; F1 profiles, 9.7%; F2 profiles, 2.8%; and RS profiles, 75.6%. These distributions are similar to that found within the dLGN in that the RS inputs (the presumed “modulators”) far outnumber the RL inputs (the presumed “drivers”). However, in comparison to the dLGN, the pulvinar nucleus receives significantly fewer numbers of RL, F1, and F2 contacts and significantly higher numbers of RS contacts. Thus, the RS/RL synapse ratio in the pulvinar nucleus is 24:1, in contrast to the 5:1 RS/RL synapse ratio in the dLGN (Van Horn et al., 2000 ). In first‐order nuclei, the lower RS/RL synapse ratio may result in the transfer of visual information that is largely unmodified. In contrast, in higher‐order nuclei, the higher RS/RL synapse ratio may allow for a finer modulation of driving inputs. J. Comp. Neurol. 454:482–494, 2002.

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.

Synaptic targets of cholinergic terminals in the pulvinar nucleus of the cat.

We compared the cholinergic innervation of the pulvinar nucleus, a thalamic association nucleus, to previous studies of the cholinergic innervation of the dorsal lateral geniculate nucleus (dLGN), a thalamic relay nucleus. Both nuclei receive a dense innervation from cholinergic cells of the brainstem parabrachial region (PBR). In the dLGN, PBR terminals are located in close proximity to retinal terminals. Our goal was to determine whether PBR terminals in the pulvinar nucleus are located in close proximity to corticothalamic terminals. We identified PBR terminals with a monoclonal antibody directed against choline acetyltransferase (ChAT). Cholinergic terminals contacted dendrites (142 of 160, or 89%) or vesicle‐filled profiles (18 of 160, or 11%). A subset of 55 terminals was stained for γ‐aminobutyric acid (GABA) to determine whether profiles postsynaptic to cholinergic terminals originate from thalamocortical cells (GABA‐) or interneurons (GABA+). The majority (44 of 55, or 80%) of postsynaptic profiles were GABA‐ dendrites. The minority (11 of 55, or 20%) were GABA+ dendrites with vesicles. This distribution of contacts is very similar to that seen in the dLGN. However, the most significant finding was that most cholinergic contacts (121 of 160, or 76%) were located within complex clusters identified as glomeruli. This is the primary site of contacts made by corticothalamic terminals originating from layer V cells. These results suggest that while the PBR enhances retinal signals in the dLGN, it may also enhance cortical signals in the pulvinar nucleus. Thus, activity in the PBR may stimulate both an increased flow of retinal information to visual cortex, as well as an increased flow of information between different visuomotor areas of cortex. J. Comp. Neurol. 387:266–278, 1997.