Esther Marije Klop

Two parts of the nucleus prepositus hypoglossi project to two different subdivisions of the dorsolateral periaqueductal gray in cat

The dorsolateral column of the mesencephalic periaqueductal gray (PAG) is a separate part of the PAG. Its afferent sources, efferent targets, and neurochemical properties differ from the adjacent PAG columns. The dorsolateral PAG is thought to be associated with aversive behaviors, but it is not yet understood how these behaviors are brought about. To elucidate the function of the PAG further, in the present study we investigated which brainstem regions project to the dorsolateral PAG. Wheat germ agglutinin‐horseradish peroxidase (WGA‐HRP) injections involving the dorsolateral PAG, but extending into the lateral part, resulted in many retrogradely labeled cells in the pontine and medullary tegmentum bilaterally. However, it was concluded that these neurons were labeled from the lateral PAG, because no anterograde labeling was found in the dorsolateral PAG after a large injection into the tegmentum. Retrogradely labeled cells were also found in the nucleus prepositus hypoglossi (PPH), mainly contralaterally. Injections of [3H]leucine or WGA‐HRP in the PPH resulted in anterogradely labeled fibers in the dorsolateral PAG. Two separate distribution patterns were found. The caudal and intermediate PPH projected to a small region on the dorsolateral edge of the dorsolateral column, whereas the supragenual PPH distributed labeled fibers to all other parts of the dorsolateral PAG, except the area on the dorsolateral edge. These separate PPH projections suggest that two subdivisions exist within the dorsolateral PAG. The present findings suggest a role for the dorsolateral PAG in the oculomotor system. J. Comp. Neurol. 492:303–322, 2005.

Direct projections from the nucleus retroambiguus to cricothyroid motoneurons in the cat

Vocalization can be elicited by stimulation in the periaqueductal gray (PAG). Light-microscopical tracing and physiological studies have revealed that the PAG uses the nucleus retroambiguus (NRA) as a relay to excite the vocalization muscle motoneurons. Direct NRA projections have been demonstrated to pharyngeal and abdominal wall muscle motoneurons, but not to laryngeal motoneurons. In two cats 0.1% cholera toxin subunit b was injected in the cricothyroid muscle of the larynx to retrogradely label its motoneurons, and 2.5% wheat germ agglutinin-horseradish peroxidase was injected into the NRA to anterogradely label its fibers. The electronmicroscopical results indicate that the NRA fibers make monosynaptic contacts with cricothyroid motoneuronal dendrites. Almost all NRA terminal profiles had asymmetrical synapses and contained mostly round or pleiomorphic vesicles, which strongly suggests that the NRA-cricothyroid motoneuronal projection is an excitatory pathway.

Lamina I-periaqueductal gray (PAG) projections represent only a limited part of the total spinal and caudal medullary input to the PAG in the cat.

The periaqueductal gray is well known for its involvement in nociception control, but it also plays an important role in the emotional motor system. To accomplish these functions the periaqueductal gray receives input from the limbic system and from the caudal brainstem and spinal cord. Earlier studies gave the impression that the majority of the periaqueductal gray projecting cells in caudal brainstem and spinal cord are located in the contralateral lamina I, which is involved in nociception. The present study in the cat, however, demonstrates that of all periaqueductal gray projecting neurons in the contralateral caudal medulla less than 7% was located in lamina I. Of the spinal periaqueductal gray projecting neurons less than 29% was located in lamina I. However, within the spinal cord large segmental differences exist: in few segments of the enlargements the lamina I-periaqueductal gray projecting neurons represent a majority. In conclusion, although the lamina I-periaqueductal gray projection is a very important nociceptive pathway, it constitutes only a limited part of the total projection from the caudal medulla and spinal cord to the periaqueductal gray. These results suggest that a large portion of the medullo- and spino-periaqueductal gray pathways conveys information other than nociception.

Segmental and laminar organization of the spinothalamic neurons in cat : Evidence for at least five separate clusters

The spinothalamic tract (STT), well known for its role in the relay of information about noxe, temperature, and crude touch, is usually associated with projections from lamina I, but spinothalamic neurons in other laminae have also been reported. In cat, no complete overview exists of the precise location and number of spinal cells that project to the thalamus. In the present study the laminar distribution of retrogradely labeled cells in all spinal segments (C1–Coc2) was investigated after large WGA‐HRP injections in the thalamus. The results show that this distribution of STT cells differed greatly between the different spinal segments. Quantitative analysis showed that there exist at least five separate clusters of spinothalamic neurons. Lamina I neurons in cluster A and lamina V neurons in cluster B are mainly found contralaterally throughout the length of the spinal cord. Cluster C neurons are located bilaterally in the ventrolateral part of laminae VI–VII and lamina VIII of the C1–C3 spinal cord. Cluster D neurons were found contralaterally in lamina VI in the C1–C2 segments, and cluster E neurons were located mainly contralaterally in the medial part of laminae VI–VII and lamina VIII of the lumbosacral cord. Most spinothalamic neurons are not located in the enlargements and most spinothalamic neurons are not located in lamina I, as suggested by several other authors. The location of the spinothalamic neurons shows remarkable similarities, but also differences, with the location of spino‐periaqueductal gray neurons. J. Comp. Neurol. 493:580–595, 2005.

C1–C3 spinal cord projections to periaqueductal gray and thalamus: A quantitative retrograde tracing study in cat

By far, the strongest spinal cord projections to periaqueductal gray (PAG) and thalamus originate from the upper three cervical segments, but their precise organization and function are not known. In the present study in cat, tracer injections in PAG or in thalamus resulted in more than 2400 labeled cells, mainly contralaterally, in the first three cervical segments (C1-C3), in a 1:4 series of sections, excluding cells in the dorsal column and lateral cervical nuclei. These cells represent about 30% of all neurons in the entire spinal cord projecting to PAG and about 45% of all spinothalamic neurons. About half of the C1-C3 PAG and C1-C3 thalamic neurons were clustered laterally in the ventral horn (C(1-3vl)), bilaterally, with a slight ipsilateral preponderance. The highest numbers of C(1-3vl)-PAG and C(1-3vl)-thalamic cells were found in C1, with the greatest density rostrocaudally in the middle part of C1. A concept is put forward that C(1-3vl) cells relay information from all levels of the cord to PAG and/or thalamus, although the processing of specific information from upper neck muscles and tendons or facet joints might also play a role.

Nucleus retroambiguus projections to the periaqueductal gray in the cat

The nucleus retroambiguus (NRA) of the caudal medulla is a relay nucleus by which neurons of the mesencephalic periaqueductal gray (PAG) reach motoneurons of pharynx, larynx, soft palate, intercostal and abdominal muscles, and several muscles of the hindlimbs. These PAG‐NRA‐motoneuronal projections are thought to play a role in survival behaviors, such as vocalization and mating behavior. In the present combined antero‐ and retrograde tracing study in the cat, we sought to determine whether the NRA, apart from the neurons projecting to motoneurons, also contains cells projecting back to the PAG. After injections of WGA‐HRP in the caudal and intermediate PAG, labeled neurons were observed in the NRA, with a slight contralateral preponderance. In contrast, after injections in the rostral PAG or adjacent deep tectal layers, no or very few labeled neurons were present in the NRA. After injection of [3H]leucine in the NRA, anterograde labeling was present in the most caudal ventrolateral and dorsolateral PAG, and slightly more rostrally in the lateral PAG, mainly contralaterally. When the [3H]leucine injection site extended medially into the medullary lateral tegmental field, labeling was found in most parts of the PAG as well as in the adjoining deep tectal layers. No labeled fibers were found in the dorsolateral PAG, and only a few were found in the rostral PAG. Because the termination pattern of the NRA fibers in the PAG overlaps with that of the sacral cord projections to the PAG, it is suggested that the NRA‐PAG projections play a role in the control of motor functions related to mating behavior. J. Comp. Neurol. 445:47–58, 2002.

Infralimbic cortex projects to all parts of the pontine and medullary lateral tegmental field in cat

The infralimbic cortex (ILc) in cat is the ventralmost part of the anterior cingulate gyrus. The ILc, together with the amygdala, bed nucleus of the stria terminalis and lateral hypothalamus, is involved in the regulation of fear behavior. The latter three structures are thought to take part in triggering the fear response by means of their projections to the pontine and medullary lateral tegmental field (LTF). The LTF is a large region extending from the parabrachial nuclei rostrally to the spinal cord caudally. It contains almost all the premotor interneurons for the brainstem and for some upper spinal cord motoneurons innervating the muscles of face, head and throat. The question is whether ILc also projects to the LTF. Such a pathway would allow the ILc to influence the fear response by acting directly on these premotor interneurons. Anterograde tracer injections were made in the medial surface of the cortex in four cats. Only when the injection sites involved ILc were anterogradely labeled fibers observed throughout the rostrocaudal extent of the LTF. To verify whether these projections indeed originated from ILc, in two other cases retrograde tracer injections were made in the pontomedullary LTF. The results showed many retrogradely labeled neurons in ILc, but none in adjacent cortical regions. These results show that the ILc projects to the LTF in cat and can possibly modulate the fear response not only via indirect but also via direct routes to the premotor interneurons in the brainstem.

Periparabigeminal and adjoining mesencephalic tegmental field projections to the dorsolateral periaqueductal grey in cat - a possible role for oculomotor input in the defensive system.

The dorsolateral column of the mesencephalic periaqueductal grey (PAGdl) differs from its adjacent columns in terms of afferent and efferent connections and the distribution pattern of different histochemical substances. Functionally, PAGdl is associated with aversive and defensive behaviours, but in an earlier study of this laboratory [E.M. Klop et al. (2005) J. Comp. Neurol., 492, 303–322], it was found that PAGdl specifically receives input from the nucleus prepositus hypoglossi, which plays a role in oculomotor control. In search for other oculomotor‐related brainstem structures projecting to PAGdl we studied the projections from the parabigeminal nucleus (PBGN) and its medially adjoining periparabigeminal area (PPBGA). In three cats, injections of wheatgerm agluttinin–horseradish peroxidase involving PAGdl did not, or to only a very limited extent, result in retrogradely labelled neurons in PBGN. When the peripheral parts of PAGdl were involved in the injection site, labelled neurons were located in PPBGA, while after an injection involving only the more central parts of PAGdl they were located in the tegmentum medial to the PPBGA. An anterograde tracing study using [3H]‐leucine and biotinylated dextran amine affirmed that neurons in PPBGA project to more peripheral parts of PAGdl, while neurons located in the tegmentum medial to PPBGA project mainly to its central parts. These results provide further evidence for the existence of two different subdivisions of PAGdl. We hypothesize that PAGdl is alerted by sudden changes in the visual field, and that the PAGdl defensive system is inhibited when these changes are caused by eye movements.

Lateral cervical nucleus projections to periaqueductal gray matter in cat

The midbrain periaqueductal gray matter (PAG) integrates the basic responses necessary for survival of individuals and species. Examples are defense behaviors such as fight, flight, and freezing, but also sexual behavior, vocalization, and micturition. To control these behaviors the PAG depends on strong input from more rostrally located limbic structures, as well as from afferent input from the lower brainstem and spinal cord. Mouton and Holstege (2000, J Comp Neurol 428:389–410) showed that there exist at least five different groups of spino‐PAG neurons, each of which is thought to subserve a specific function. The lateral cervical nucleus (LCN) in the upper cervical cord is not among these five groups. The LCN relays information from hair receptors and noxious information and projects strongly to the contralateral ventroposterior and posterior regions of thalamus and to intermediate and deep tectal layers. The question is whether the LCN also projects to the PAG. The present study in cat, using retrograde and anterograde tracing techniques, showed that neurons located in the lateral two‐thirds of the LCN send fibers to the lateral part of the PAG, predominantly at rostrocaudal levels A0.6–P0.2. This part of the PAG is known to be involved in flight behavior. A concept is put forward according to which the LCN‐PAG pathway alerts the animal about the presence of cutaneous stimuli that might represent danger, necessitating flight. J. Comp. Neurol. 471:434–445, 2004.

Less than 15% of the spinothalamic fibers originate from neurons in lamina I in cat

Lamina I neurons sending their axons into the spinothalamic tract are thought to play a crucial role in nociception, but many spinothalamic fibers do not originate from lamina I neurons. In cat, no consensus exists about what percentage of the spinothalamic tract cells are located in lamina I. After wheat germ agglutinin-conjugated horseradish peroxidase injections that covered large parts of the thalamus, retrogradely labeled cells were plotted and counted in all segments of the spinal cord. Results show that, averaged over all spinal segments, the percentage of labeled lamina I neurons was 4.9-14.2%. These results demonstrate that, in contrast to what is concluded in several previous studies, lamina I in the cat provides only a limited part of the total spinal input to the thalamus.