Jane R. Clements

Localization of glutamate, glutaminase, aspartate and aspartate aminotransferase in the rat midbrain periaqueductal gray

SummaryGlutamate and aspartate are putative excitatory neurotransmitters in the central nervous system. The present study utilized novel monoclonal antibodies against fixative-modified glutamate and aspartate and polyclonal antisera against the amino acid synthesizing enzymes, glutaminase and aspartate aminotransferase, to analyze the distribution of these amino acids in the rodent midbrain periaqueductal gray. Glutamate-, aspartate-, glutaminase- and aspartate aminotransferase-like immunoreactive neurons, fibers and processes are present throughout the rostrocaudal length of the periaqueductal gray. Glutamate- and glutaminase-like immunoreactive neurons displayed a similar homogeneous pattern of distribution, being localized predominantly to the lateral and dorsal subdivisions of the periaqueductal gray. Co-localization experiments suggest that glutamate and glutaminase are in fact co-contained within the same PAG neurons. Aspartate aminotransferase-like immunoreactive neurons were distributed in a pattern similar to glutamate and glutaminase with the exception that fewer cells were stained in the dorsocaudal and the rostral third of the PAG. Aspartate-like immunoreactive neurons were less numerous than glutamate-like immunoreactive cells and were located in the lateral aspect of the PAG. These results demonstrate a specific and distinct distribution of glutamate and aspartate immunoreactive neurons and support recent data suggesting that glutamate and aspartate serve as excitatory neurotransmitters in the PAG.

Localization of glutamate in trigeminothalamic projection neurons: A combined retrograde transport-immunohistochemical study

Trigeminothalamic projection neurons are important components of the pathways for conscious perception of pain, temperature, and tactile sensation from the orofacial region. The neurotransmitters utilized by trigeminal neurons projecting to the thalamus are unknown. By use of a monoclonal antibody specific for fixative-modified glutamate and a polyclonal antiserum against glutaminase, we recently identified neurons in the trigeminal sensory complex that contain glutamate-like immunoreactivity (Glu-LI) and glutaminase-like immunoreactivity. In the present study, we utilized combined retrograde transport-immunohistochemical techniques to localize putative glutamatergic trigeminothalamic neurons. Following injection of the retrograde tracer, wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP), into the ventroposterior medial thalamus (VPM), the number of neuronal profiles that were double-labeled with WGA:HRP and Glu-LI was greatest in principal sensory nucleus (Pr5), followed by subnuclei interpolaris (Sp5I) and caudalis (Sp5C). The average percentages of projection neurons double-labeled with Glu-LI were approximately 60-70% in Pr5 and Sp5I and 40% in Sp5C. The majority of double-labeled profiles in Sp5C were located in the magnocellular layer, as opposed to the marginal and substantia gelatinosa layers. A large injection site that spread into the intralaminar thalamic nuclei and nucleus submedius--areas implicated in the processing of nociceptive information--resulted in an increase in the ratio of single-labeled to double-labeled projection profiles in Sp5C. These results suggest that glutamate may be the neurotransmitter for a majority of trigeminothalamic projection neurons located in Sp5I and Pr5. However, on the basis of anatomical association, glutamate does not appear to be the major transmitter for neurons in Sp5C that forward nociceptive information to the thalamus.

The nuclei of origin of brainstem enkephalin and cholecystokinin projections to the spinal trigeminal nucleus of the rat

The sites of origin of brain stem enkephalin and cholecystokinin projections to the rodent spinal trigeminal nucleus were studied utilizing the combined retrograde transport-peroxidase antiperoxidase immunohistochemical technique. Several brain stem areas were found to contain enkephalin-like immunoreactive double-labeled neurons following injection of wheat germ agglutinin-horseradish peroxidase or horseradish peroxidase into the spinal trigeminal nucleus. The largest numbers of enkephalin double-labeled neurons were identified in the nucleus pontis oralis, nucleus raphe medianis, medial vestibular nucleus and the midbrain periaqueductal gray. Enkephalin projections to the spinal trigeminal nucleus were also found to originate from the nucleus solitarius, nucleus raphe pallidus, nucleus raphe magnus, nucleus raphe dorsalis, nucleus reticularis paragigantocellularis, nucleus reticularis gigantocellularis pars alpha and the deep mesencephalic nucleus. In contrast to the numerous sources of enkephalin input to the spinal trigeminal nucleus, cholecystokinin projections to this region were limited to four brain stem nuclei. These included the nucleus solitarius, raphe obscurus, nucleus paragigantocellularis and the ventral reticular nucleus of the medulla. The finding that only a small number of brain stem cholecystokinin-like immunoreactive neurons project to the spinal trigeminal nucleus supports the hypothesis that most of the cholecystokinin input to the spinal trigeminal nucleus arises from primary afferent trigeminal fibers. The spinal trigeminal nucleus is known to play a role in processing sensory information and in the transmission of orofacial nociception. The present study identifies several brain stem sites which provide enkephalin and/or cholecystokinin input to the spinal trigeminal nucleus. Several of these nuclei have been implicated as components of the endogenous pain control system and the present results raise the possibility that they may modulate incoming orofacial nociception by releasing the endogenous opioid, enkephalin. Cholecystokinin, on the other hand, has been demonstrated in other studies to attenuate the action of opiates and thus may play an opposing role in the spinal trigeminal nucleus.

An ultrastructural description of glutamate-like immunoreactivity in the rat cerebellar cortex

This study provides the first ultrastructural description of glutamate-like immunoreactive neurons and processes in the cerebellar cortex of the rat. Glutamate-like immunoreactivity was seen in parallel fibers, granule cell perikarya, Purkinje cell dendrites, and mossy fiber glomeruli. These data support previous studies that have suggested that granule cells may use glutamate as a neurotransmitter. However, not all granule cells or all parallel fibers were glutamate-like immunoreactive, suggesting that some granule cells may use transmitters other than glutamate. The presence of glutamate-like immunoreactivity in mossy fibers supports the hypothesis that some cerebellar afferent systems may use glutamate as a neurotransmitter.

Immunocytochemical localization of glutamate dehydrogenase in mitochondria of the cerebellum: an ultrastructural study using a monoclonal antibody

Monoclonal antibodies to glutamate dehydrogenase (GDH) were produced and shown to have high degrees of specificity using immunoblots and ELISA. Immunocytochemical staining of electron microscopic preparations revealed selective intense staining of mitochondria in Bergmann glia, oligodendrocytes and astrocytes in the cerebellum of the rat. Differential intensity of staining among mitochondria within individual glial cells and between glial cells was observed and may provide an anatomical means of detecting differences in glutamate metabolism.

The effects of different pretreatment conditions and fixation regimes on serotonin immunoreactivity: a quantitative light microscopic study.

An investigation was designed to evaluate the effects of three different fixation regimes on the retention of serotonin-like immunoreactivity in rat midbrain tissue sections. The effects of pretreatment with pargyline-HCl and l-tryptophan on the volume fraction of serotonin-like immunoreactive processes were also examined. Rat brain tissue was fixed with 4% paraformaldehyde (Pf), 4% paraformaldehyde-0.2% picric acid-0.05% glutaraldehyde (Pf-Pa-G), or 4% paraformaldehyde-0.2% glutaraldehyde (Pf-G). Tissue was subsequently processed for immunohistochemistry using a modified peroxidase-antiperoxidase technique and quantified at the light microscopic level by point counting. Fixation with Pf resulted in higher volume fraction determinations of axonal serotonin immunoreactivity than did fixation with Pf-Pa-G or Pf-G. These results provide quantitative data which indicate that even low levels of glutaraldehyde in the fixative significantly decrease serotonin immunoreactivity. Pretreatment with pargyline and tryptophan increased the amount of serotonin immunoreactivity in tissue fixed with Pf-G but not in tissue fixed with Pf. Pretreatment with pargyline and tryptophan is thus recommended when using glutaraldehyde in the fixation process to assure adequate serotonin immunoreactivity. Pretreatment in conjunction with glutaraldehyde fixation, however, appears to cause differential increases in serotonin-like immunoreactivity within brain nuclei that may compromise the interpretation of results.

A quantitative light microscopic analysis and ultrastructural description of cholecystokinin-like immunoreactivity in the spinal trigeminal nucleus of the rat

The spinal trigeminal nucleus is involved in orofacial sensory transmission. Cholecystokinin octapeptide has been identified in axons in this nucleus and appears to play a role in the transmission of orofacial sensation from the trigeminal ganglia to the spinal trigeminal nucleus. Although cholecystokinin has been reported in axonal processes within the spinal trigeminal nucleus at the light microscopic level, nothing is known about the synaptic relationships of these cholecystokinin axons. The goals of this study were to quantitatively determine the volume fraction of cholecystokinin-like immunoreactive cell bodies and fibers in the three subnuclei of the spinal trigeminal nucleus, to provide the first ultrastructural description of cholecystokinin-like immunoreactive processes within these subnuclei and to analyse the synaptic relationships of cholecystokinin-like immunoreactive processes within the spinal trigeminal nucleus neuropil. Cholecystokinin-like immunoreactivity was localized by the peroxidase-antiperoxidase method or the peroxidase labeled, avidin-biotin technique and quantified at the light microscopic level by point counting. Immunoreactive fibers were present in all three subnuclei, but the greatest volume fraction of immunoreactive axons was obtained in laminae I and II of the nucleus caudalis. No immunoreactive cell bodies were evident in any of the subnuclei. The majority of immunoreactive profiles in all three subnuclei were identified ultrastructurally as axon terminals that contained both small and medium sized agranular vesicles and infrequently, large dense core vesicles. These immunoreactive terminals were usually found in close contact with non-immunoreactive dendrites with which they were observed to form asymmetric synapses. Immunoreactive terminals were occasionally observed to contact the cell bodies of large non-immunoreactive neurons on the border of laminae I and II in the nucleus caudalis. These results indicate that cholecystokinin-like immunoreactive processes are present throughout the spinal trigeminal nucleus, and in nucleus caudalis show a distribution similar to that reported for the spinal cord dorsal horn. Immunoreactive axons make synaptic contact with both the dendrites and perikarya of spinal trigeminal nucleus neurons. No axoaxonic synapses were observed. These findings suggest that cholecystokinin plays an important role in spinal trigeminal nucleus function. The possible colocalization of cholecystokinin and substance P in the spinal trigeminal nucleus, and the possible role of cholecystokinin in attenuating the action of opioids in the spinal trigeminal nucleus are also discussed.