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The development of selective 5-HT histofluorescent and immunohistochemical techniques has allowed the localisation of 5-HT-containing cell bodies and nerve terminals in the CNS. In 1964, Dahlstrom and Fuxe used a technique o f treating brain slices with formaldehyde to induce fluorescence specifically in monoamines to label 5-HT-containing neurones and nerve terminals in the rat brain. They described nine different groups of 5-HT-containing neurones which were located within the brainstem near to the midline (figure 1.3). The clusters

Figure 1.3

A sagittal section through the rat brainstem illustrating the position of the different groups o f 5-HT-containing neurones according to the w ork of Dahlstrom and Fuxe (1964) except that in this diagram the group o f 5-HT neurones in the area postrema are labelled B4.

B l; Raphe pallidus nucleus, B2; Raphe obscurus nucleus, B3; Raphe magnus nucleus, B4; area postrema, B5; Median raphe nucleus, B6 and B7; Dorsal raphe nucleus, B8; Caudal linear nucleus, B9; Supralemniscal region.

of neurones were labelled B1 to B9 in a caudalrostral direction. Furthermore, the groups of 5-HT-containing neurones could be divided into caudal and rostral subdivisions. The caudal division was comprised of 5-HT-containing neurones in the raphe pallidus nucleus (B l), the raphe obscurus nucleus (B2 and B4), the raphe magus nucleus (B3) and the ventrolateral medulla (B4) whereas the rostral division was composed of neurones in the median raphe nucleus (B5 and B8), the dorsal raphe nucleus (B6 and B8), caudal linear nucleus (B8) and the supralemniscal regions (B9). The development of m ore sensitive immunocytochemical techniques has led to the discovery of other 5-HT- containing neurones, in particular, clusters of small-sized cells located in the medial NTS and area postrema (Steinbusch, 1981).

1.9a Ascending projections of 5-HT-containing neurones

The ascending axons of rat 5-HT-containing neurones have been shown by HRP retrograde tracing to project to the forebrain via the medial forebrain bundle (Takagi, Shiosaka, Toyyama, Senba and Sakanaka, 1980). An immunocytochemical study using antiserum raised against 5-HT was shown to bind to fibres in the thalamus, in particular, the periventricular nucleus o f the thalamus (Cropper, Eisenman and Azitia, 1984). A study mapping nerve degeneration following lesions in the median and dorsal raphe showed projections from these nuclei to the hypothalamus, the thalamus, the amygdala and olfactory tubercle (Conrad, Leonard and Pfaff, 1974). These projections were confirmed using anterograde tracing by injecting pH]-proline into the raphe nuclei. Kohler and Steinbusch (1981) injected the retrograde Buoresence tracers Granular blue or Propidium into the the entorhinal cortex or the hippocampus and then double-labelled neurones using an antisera for 5-HT. 5- HT-containing neurones projecting to the entorhinal cortex and the hippocampus were found in medial and dorsal raphe nuclei. An

iimnimohistochemical study which traced the projections of 5-HT-containing neurones in the medial, dorsal and B9 regions showed that these areas terminate in the neocortical and cingulate cortical regions and layers I and IV o f the cerebral cortex (Lidov, Grzanna and Molliver, 1980).

1.9b Descending projections of 5-HT-containing neurones

The descending projections from 5-HT-containing neurones innervate the spinal cord in a highly organised manner. Lamina I of the dorsal horn receives a dense innervation from neurones in the raphe magnus nucleus which project to the spinal cord through the dorsolateral fasiciculus (Steinbusch, 1981). HRP injections into the T1 and T2 levels of the spinal cord combined with 5-HT- immunohistochemistry showed 5-HT-containing neurones in the raphe pallidus and raphe obscurus (Loewy and McKellar, 1981). Injections of pH]-leucine, pH]-lysine and pH]-proline iuto both these raphe nuclei demonstrated anterograde projections to the intermediolateral cell column of the spinal cord which contains sympathetic preganglionic neurones and the ventral horn of the spinal cord. Microinjections of the retrograde tracer fast blue into the diaphragm of cats showed labelling in the phrenic m otor nucleus which was also shown to contain dense levels of 5-HT-immunoreactive fibres and varicosities in close proximity to phrenic motor neurones (Holtman, Norman, Skirboll, Dretchen, Cuello, Visser, Hokfelt and Gillis, 1984). This suggests that 5-HT-containing neurones are involved in the central control of respiration.

Even though there are certain "hot-spots" of innervation from 5-HT-containing neurones as described above, lower but significant levels of 5-HT- immunoreactive fibres were observed in almost every area of the brain except the m ajor fibre tracts such as the corpus callosum and optic tract (Jacobs and Azmitia, 1992).

1.9c Co-localisation of neurochemicals with 5-HT-containing neurones

A num ber of neurochemicals have been suggested to be colocalised in 5-HT- containing cell bodies. An immunocytochemical study in the rat using antibodies to 5-HT, substance P (SP) and TRH showed the presence o f all three neurochemicals within neurones in the regions of the raphe pallidus and raphe obscurus (Johasson, Hokfelt, Pemow, Jeffcoate, White, Steinbusch, Verhofstad, Emson and Spindel, 1981). In the rat dorsal raphe, an immunohistochemical study has shown that the number of neurones expressing the G ABA synthesising enzyme glutamate decarboxylase is reduced with prior treatment of the selective 5-HT neurotoxin 5,7-dihydroxytryptamine (Nanopoulos, Belin, M aitre and Pujol, 1982). This indicates that 5-HT and G ABA are co-localised in neurones in the dorsal raphe. In the rat, injections of true blue into the parabrachial nucleus retrogradely labelled neurones in the area postrema which were immunoreactive for 5-HT and tyrosine hydroxylase suggesting that 5-HT and noradrenaline can be co-localised within neurones of the area postrema (Miceli, Post and Van de Kooy, 1987).

1.10 5-HT innervation of the DMN

Immunocytochemical studies in the rat have shown a high density of 5-HT- containing nerve fibres and nerve terminals in the DM N (Steinbusch, 1981). The origin of this 5-HT innervation is unclear although retrograde tracer studies using HRP and rhodamine beads to map afferent inputs to the DM N showed projections from neurones in the caudal raphe nuclei, in particular, the nucleus raphe obscurus and the nucleus raphe pallidus (Rogers et. al., 1980; Thor and Helke, 1987). These nuclei, located in the midline area of the brainstem, are regarded as being predominantly composed of 5-HT-containing neurones and have been demonstrated to contain neurones with 5-HT immunoreactivity which project to the DMN (Thor and Helke, 1987). Vagal afferents innervating the

DM N also express 5-HT imimmoreactivity providing evidence that 5-HT may be involved in controlling vago-vagal interactions (Sykes et. al., 1994). Injections o f PRV into the pancreas of the rat retrogradely labelled DVMs in the DM N and transneuronally labelled neurones throughout the medulla oblongata (Loewy et. at., 1994). The nucleus raphe magnus, obscurus and pallidus all contained neurones with 5-HT immunoreactivity indicating that these areas provide a 5-HT innervation to the DMN. In the cat, electrical stimulation o f the nucleus raphe obscurus induced the release o f 5-HT in the NTS and DM N which was measured using micro-dialysis techniques (Brodin, Linderoth, Goiny, Yamamoto, Gazelius, M illhom , Hokfelt and Ungerstedt, 1990). These results suggest that the 5-HT innervation of the DM N may be from 5-HT-containing neurones located in midline raphe nuclei possibly the nucleus raphe obscurus and nucleus raphe pallidus.