Specific Neurochemical Derangements of Brain Projecting Neurons in Apolipoprotein E-Deficient Mice

Abstract: Apolipoprotein E (apoE)-deficient mice provide a useful system for studying the role of apoE in neuronal maintenance and repair. Previous studies revealed specific memory impairments in these mice that are associated with presynaptic derangements in projecting forebrain cholinergic neurons. In the present study we examined whether dopaminergic, noradrenergic, and serotonergic projecting pathways of apoE-deficient mice are also affected and investigated the mechanisms that render them susceptible. The densities of nerve terminals of forebrain cholinergic projections were monitored histochemically by measurements of acetylcholinesterase activity, whereas those of the dopaminergic nigrostriatal pathway, the noradrenergic locus coeruleus cortical projection, and the raphe-cortical serotonergic tract were measured autoradiographically using radioligands that bind specifically to the respective presynaptic transporters of these neuronal tracts. The results obtained revealed that synaptic densities of cholinergic, noradrenergic, and serotonergic projections in specific brain regions of apoE-deficient mice are markedly lower than those of controls. Furthermore, the extent of presynaptic derangement within each of these tracts was found to be more pronounced the further away the nerve terminal is from its cell body. In contrast, the nerve terminal density of the dopaminergic neurons that project from the substantia nigra to the striatum was unaffected and was similar to that of the controls. The rank order of these presynaptic derangements at comparable distances from the respective cell bodies was found to be septohippocampal cholinergic > nucleus basalis cholinergic > locus coeruleus adrenergic > raphe serotonergic ? nigrostriatal dopaminergic, which interestingly is similar to that observed in Alzheimer's disease. These results suggest that two complementary factors determine the susceptibility of brain projecting neurons to apoE deficiency: pathway-specific differences and the distance of the nerve terminals from their cell body.     

Effects of estrogens on cholinergic neurons in the rat basal nucleus

Estrogen replacement in postmenopausal women may help prevent or delay development of Alzheimer's disease. Because loss of basal forebrain cholinergic neurons with reductions in choline acetyltransferase (ChAT) concentration are associated with Alzheimer's disease, we investigated the effect of estradiol (E(2)) and J 861, a non-feminizing estrogen, on cholinergic neurons in the basal forebrain. Ovariectomized rats received E(2), J 861 or vehicle, and basal forebrain sections through the substantia innominata, medial septum, and nucleus of the diagonal band were immunostained for ChAT. ChAT-immunoreactive cells in the basal forebrain were significantly reduced in the ovariectomized rats compared to intact rats, but those ovariectomized rats receiving estrogen replacement with E(2) and J 861 had near normal levels of ChAT-positive neurons. While retrograde tracing experiments with fluorogold injected into the prefrontal cortex showed no significant differences in the number of fluorogold-labeled cells among the groups, ChAT-immunoreactive cells and double-labeled cells were significantly lower in OVX rats than in intact and E(2) rats. Some substantia innominata cells in the J 861 rats were ChAT/estrogen receptor alpha-positive. These results suggest that E(2) and J 861 have positive effects on cholinergic neurons that project from the basal nucleus to the forebrain cortex.     

Catechol-O-Methyltransferase (COMT) Protein Expression and Activity after Dopaminergic and Noradrenergic Lesions of the Rat Brain

The occurrence of catechol-O-methyltransferase (COMT) in presynaptic neurons remains controversial. This study utilized dopaminergic and noradrenergic toxins to assess the presence of COMT in the presynaptic neurons originating from the substantia nigra, ventral tegmental area or locus coeruleus. Destruction of dopaminergic and noradrenergic neurons was assessed by measuring the dopamine and noradrenaline content in the projection areas of these neurons. Additionally, COMT protein expression and activity were examined in several projection areas to determine whether there are any changes in COMT values. Colocalization studies were done to identify COMT-containing postsynaptic neurons. Despite successful lesioning of dopaminergic and noradrenergic neurons, no changes in COMT protein expression or activity could be noted. These results strongly suggest that COMT is not present in presynaptic dopaminergic and noradrenergic neurons. There was a high colocalization of COMT with the GABAergic marker of short neurons both in the striatum and cortex but only a weak, if any, with the cholinergic marker in the cortex.     

Choline acetyltransferase activity at different ages in brain of Ts65Dn mice, an animal model for Down's syndrome and related neurodegenerative diseases

Ts65Dn mice, trisomic for a portion of chromosome 16 segmentally homologous to human chromosome 21, are an animal model for Down's syndrome and related neurodegenerative diseases, such as dementia of the Alzheimer type. In these mice, cognitive deficits and alterations in number of basal forebrain cholinergic neurons have been described. We have measured in Ts65Dn mice the catalytic activity of the cholinergic marker, choline acetyltransferase (ChAT), as well as the activity of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), in the hippocampus and in cortical targets of basal forebrain cholinergic neurons. In mice aged 10 months, ChAT activity was significantly higher in Ts65Dn mice, compared to 2N animals, in the hippocampus, olfactory bulb, olfactory cortex, pre-frontal cortex, but not in other neocortical regions. At 19 months of age, on the other hand, no differences in ChAT activity were found. Thus, alterations of ChAT activity in these forebrain areas seem to recapitulate those recently described in patients scored as cases of mild cognitive impairment or mild Alzheimer's disease. Other neurochemical markers putatively associated with the disease progression, such as those implicating astrocytic hyperactivity and overproduction of amyloid precursor protein family, were preferentially found altered in some brain regions at the oldest age examined (19 months).     

Changes in activity and expression of phosphofructokinase in different rat brain regions after basal forebrain cholinergic lesion

Selective lesion of rat basal forebrain by the cholinergic immunotoxin 192IgG-saporin was used as an animal model to address the question of whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease may be related to impaired cholinergic transmission. At different times after creating the immunolesion, the isoenzyme pattern and steady-state mRNA levels of the key glycolytic enzyme phosphofructokinase were determined in cortex, hippocampus, basal forebrain and nucleus caudatus. The loss of cholinergic input was accompanied by a persistent decrease in choline acetytransferase and acetylcholine esterase activities in the cortical target areas similar to the cholinergic malfunction seen in Alzheimer's dementia. The basal forebrain lesion induced by the immunotoxin resulted in a transient increase in phosphofructokinase activity peaking on day 7 after inducing the lesion in cortical areas. In parallel, an increased steady-state level of phosphofructokinase mRNA was determined by RT/real-time PCR and in situ hybridization. In contrast, analysis by western blotting and quantitative PCR revealed no changes in the phosphofructokinase isoenzyme pattern after immunolesion. It is concluded that common metabolic mechanisms may underlie the degenerative and repair processes in denervated rat brain and in the diseased Alzheimer's brain.     

Afferent projections to the locus coeruleus nucleus in the cat. Study by the horseradish peroxidase technic]

Using a retrograde tracer technique with horseradish peroxidase, we have revealed some afferent projections to the locus coeruleus complex from the contralateral pontine tegmentum, raphe nuclei, substantia nigra, nucleus of the solitory tract, dorsal motor nucleus of the vagus and other regions of the ponto-bulbar reticular formation as well as from hypothalamic and preoptic areas.     

Is the neuronal basis of Alzheimer's disease cholinergic or glutamatergic?

The hypothesis that the symptomatology of Alzheimer's disease is attributable to cholinergic dysfunction is supported by postmortem studies that have demonstrated reduced choline acetyltransferase (ChAT) activity across all areas of cerebral cortex and diminished numbers of perikarya in the basal forebrain nucleus basalis of Meynert. Biopsy studies of ChAT activity, choline uptake, and acetylcholine synthesis also suggest that cholinergic denervation occurs relatively early in the course of the disease, and in confirmation of postmortem data, correlates with the severity of cognitive impairment. An alternative hypothesis to explain the dementia of Alzheimer's disease is the glutamatergic hypothesis. This is based largely on postmortem evidence indicating reduced binding and uptake of D[3H]aspartate, as well as loss of a number of other putative markers, such as phosphate-activated glutaminase activity, glutamate concentration, and the number of pyramidal cell perikarya, with this latter change correlating with the severity of dementia. Short-comings of each hypothesis are discussed and the merits of single neuron hypotheses to explain the dementia of Alzheimer's disease are considered.     

Immunohistochemical mapping of galanin-like neurons in the rat central nervous system

Using an antiserum generated in rabbits against synthetic galanin (GA) and the indirect immunofluorescence method, the distribution of GA-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system (CNS) and a detailed stereotaxic atlas of GA-like neurons was prepared. GA-like immunoreactivity was widely distributed in the rat CNS. Appreciable numbers of GA-positive cell bodies were observed in the rostral cingulate and medial prefrontal cortex, the nucleus interstitialis striae terminalis, the caudate, medial preoptic, preoptic periventricular, and preoptic suprachiasmatic nuclei, the medial forebrain bundle, the supraoptic, the hypothalamic periventricular, the paraventricular, the arcuate, dorsomedial, perifornical, thalamic periventricular, anterior dorsal and lateral thalamic nuclei, medial and central amygdaloid nuclei, dorsal and ventral premamillary nuclei, at the base of the hypothalamus, in the central gray matter, the hippocampus, the dorsal and caudoventral raphe nuclei, the interpeduncular nucleus, the locus coeruleus, ventral parabrachial, solitarii and commissuralis nuclei, in the A1, C1 and A4 catechaolamine areas, the posterior area postrema and the trigeminal and dorsal root ganglia. Fibers were generally seen where cell bodies were observed. Very dense fiber bundles were noted in the septohypothalamic tract, the preoptic area, in the hypothalamus, the habenula and the thalamic periventricular nucleus, in the ventral hippocampus, parts of the reticular formation, in the locus coeruleus, the dorsal parabrachial area, the nucleus and tract of the spinal trigeminal area and the substantia gelatinosa, the superficial layers of the spinal cord and the posterior lobe of the pituitary. The localization of the GA-like immunoreactivity in the locus coeruleus suggests a partial coexistence with catecholaminergic neurons as well as a possible involvement of the GA-like peptide in a neuroregulatory role.     

Opiocortin and catecholamine projections to raphe nuclei

Afferent projections to the nucleus raphe magnus (NRM) and dorsal raphe nucleus (DRN) were identified using retrograde transport of horseradish peroxidase conjugated wheat germ agglutinin (HRP-WGA). Neurons were labeled in important nociceptive regions including periaqueductal gray (PAG), arcuate nucleus, lateral hypothalamus and medial thalamic nuclei following both injections. We have immunocytochemically identified opiocortin/WGA neurons in the arcuate nucleus following NRM and DRN injections. Dual stained catecholamine/WGA perikarya were found in zona incerta, locus coeruleus, substantia nigra, nucleus tractus solitarius and adjacent A2, C2 and C3, lateral paragigantocellular reticular nucleus/C1 and lateral reticular nucleus/A1 following DRN injections and in zona incerta, substantia nigra, nucleus tractus solitarius/A2 and lateral reticular nucleus/A1 after NRM injections. These results provide further evidence for opiocortin and catecholamine modulation of analgesia.     

Possible destruction of cholinoceptive neurons by overstimulation of cholinergic tracts

It is well established that intracerebral injections of kainic acid may cause not only neuronal cell destruction at the injection site, but also losses in some distant regions. The mechanisms are different. The distant, but not the local, destruction can be produced by folic as well as by kainic acid and prevented by pretreatment of the animal with diazepam. Overexcitation of excitatory projections is believed responsible for the distant damage and evidence is presented that in some instances the projections involved are cholinergic. Thus, for example, injections of kainic acid or folic acid into the substantia innominata of rats destroy neurons in areas such as the pyriform cortex and amygdala which receive cholinergic projections from the injected area. Some of the destroyed neurons are GABAergic. That the distant toxicity in these areas can be partially blocked by scopolamine and is accompanied by decreases in the number of muscarinic binding sites is consistent with a cholinergic mechanism. Distant damage also occurs in the thalamus but this appears to be mediated by a noncholinergic projection. Similar injections of folic acid or kainic acid into the rostral pontine tegmentum, another area with cholinergic cells, cause destruction of both dopaminergic and GABAergic neurons in the substantia nigra. The effect on the GABAergic but not that on the dopaminergic cells is blocked by scopolamine. The results are discussed in relation to possible mechanisms of epilepsy and of selective neuronal losses in diseases such as Parkinson's disease.     

Changes of Biogenic Amines and Their Metabolites in Postmortem Brains from Patients with Alzheimer-Type Dementia

Noradrenaline (NA), 3,4-dihydroxyphenylethylamine (dopamine, DA), 5-hydroxytryptamine (serotonin, 5-HT), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) were measured in 22 regions of postmortem brains from four histologically verified cases with Alzheimer-type dementia (ATD) and nine histologically normal controls. Compared with the controls, concentrations of 5-HT and 5-HIAA in the ATD brains were significantly reduced in nine regions (superior frontal gyrus, insula, cingulate gyrus, amygdala, putamen, medial and lateral segments of globus pallidus, substantia nigra, lateral nucleus of thalamus) and in eight regions (amygdala, substantia innominata, caudate, putamen, medial and lateral segments of globus pallidus, medial and lateral nuclei of thalamus), respectively. NA concentrations of the ATD brains were significantly reduced in six regions (cingulate gyrus, substantia innominata, putamen, hypothalamus, medial nucleus of thalamus, raphe area). In contrast, significant reductions of DA and HVA concentrations in the ATD brains were found only in putamen and amygdala, respectively. The 5-HIAA/5-HT ratio in the ATD brains decreased significantly in locus coeruleus, while the HVA/DA ratio increased significantly in putamen and medial segment of globus pallidus. These findings suggest that the serotonergic and noradrenergic systems are affected, while the dopaminergic system is relatively unaffected in ATD brains.     

Retrograde degeneration of basal forebrain cholinergic neurons after neurotoxin lesions of the neocortex: application of ganglioside GM1

Injections of the neurotoxin kainic acid were made unilaterally at multiple loci in the cerebral cortex of the rat in an attempt to reproduce aspects of the central pathology of Alzheimer's disease. Neurochemical markers of cholinergic and GABAergic function in the cortex and basal forebrain, determined after various intervals, suggested that subsequent to initial destruction of cortical neuronal cell bodies, trans-synaptic retrograde degeneration of cholinergic neurons occurred in the nucleus basalis magnocellularis (NBM) projecting to the cortex.

Contrary to the situation noted after devascularizing cortical lesions, there was no spontaneous recovery from this effect of kainic acid in the ipsilateral NBM. Similarly, these retrograde effects could not be prevented by the administration of the ganglioside GM₁. These observations suggest that kainic acid compromises the plastic capacity of this cholinergic projection, perhaps by affecting the production of endogenous trophic factors. This may be of relevance in developing the use of neurotoxins for models of neurodegenerative disease.     

Localization of DJ-1 protein in the murine brain

Mutations in the DJ-1 gene have been identified to cause Parkinson's disease. In humans, nonmutated DJ-1 is expressed in specific brain areas but seems to be expressed by astrocytes rather than by neurons. In contrast, DJ-1 mRNA is mainly found in neurons in the mouse brain. We have investigated the distribution of DJ-1 protein in the mouse brain and found that DJ-1 protein is predominantly expressed by neurons but can also be detected in astrocytes. Consistent with a global role of DJ-1 in the brain, we found immunoreactivity, for example, in cortical areas, hippocampus, basolateral amygdala, the reticular nucleus of the thalamus, zona incerta, and locus coeruleus. Within the substantia nigra, however, DJ-1 is localized in both neuronal and nonneuronal cells, suggesting a distinct role in this area.     

Distribution of arginine vasotocin in the brain of the lizard Anolis carolinensis

Summary The distribution of immunoreactive arginine vasotocin (AVT-ir) was determined in the brain of the lizard Anolis carolinensis. Cells and fibers containing AVT-ir were found in the medial septal region, lamina terminalis, lateral forebrain bundle, preoptic area, supraoptic nucleus, anterior hypothalamus, paraventricular nucleus, periventricular nucleus, arcuate nucleus, and ventromedial nucleus of the thalamus. Occasional AVT-ir cells were found in the interpeduncular nucleus. Fibers containing AVT-ir were found in the cortex, around the olfactory ventricle, in the diagonal band of Broca, amygdala area, dorsal ventricular ridge, striatum, nucleus accumbens, septum, ventromedial hypothalamus, lateral hypothalamus, medial forebrain bundle, median eminence, pars nervosa, nucleus of the solitary tract, locus coeruleus, cerebellar cortex (granular layer), dorsal part of the nucleus of the lateral lemniscus, substantia nigra, and myelencephalon. The intensity of AVT-ir staining was, in general, greater in males than in females. Comparison of AVT-ir distribution in A. carolinensis with those previously published for other reptilian species revealed species-specific differences in distribution of AVT.     

Evidence for a novel pituitary protein (7B2) in human brain, cerebrospinal fluid and plasma: brain concentrations in controls and patients with Alzheimer's disease

A novel pituitary protein, designated as 7B2, recently purified in our laboratory was measured using a specific radioimmunoassay in conjunction with immuno-affinity extraction, in cerebrospinal fluid (CSF) and in plasma obtained from normal volunteers. The mean concentrations of immunoreactive (IR)-7B2 were 2154 pg/ml in CSF and 29 pg/ml in plasma. Studies by SDS-poly-acrylamide gel electrophoresis revealed that both CSF IR-7B2 and plasma IR-7B2 have an apparent molecular weight of around 20,000-21,000 as previously observed in various rat tissues. IR-7B2 was also measured in various brain regions obtained from control subjects and patients with Alzheimer's disease. IR-7B2 was widely distributed in the human brain, with the highest concentrations in substantia nigra and caudate. IR-7B2 brain concentrations were found to be similar between control subjects and patients with Alzheimer's disease. Gel permeation chromatography of extracts of various brain regions revealed two major peaks with apparent molecular weights of 45,000-50,000 and 11,000-16,000 in hypothalamus, caudate, frontal cortex, hippocampus, putamen and locus coeruleus, and only one peak with an apparent molecular weight of 14,000-16,000 in substantia nigra and globus pallidus. These data suggest that this novel pituitary protein may play a role of consequence perhaps as a neurotransmitter or as a neuromodulator in the human central nervous system.     

Spatial analysis of the neuronal density of aminergic brainstem nuclei in primary neurodegenerative and vascular dementia: a comparative immunocytochemical and quantitative study using a graph method.

A graph method was employed to analyse spatial neuronal patterns of pontine nuclei with ascending aminergic projections to the forebrain (nucleus centralis superior (NCS), raphes dorsalis (NRD) and locus coeruleus (LC)), in Alzheimer disease (AD), Huntington disease (HD), and vascular (VD) as well as "mixed-type" (VA) dementia, compared with non-demented controls (CO) and a small sample of brains from schizophrenics ("dementia praecox" (DP)). The quantitative evaluations by the "minimal spanning tree (MST)" were complemented by rough neurofibrillary tangle (NFT) counts and by semiquantitative immunohistochemical assessment of amyloid deposition, neuritic plaque formation, and cellular gliosis. The AD cases showed a significant decline of neuronal density in all nuclei examined, as compared with controls and DP. Neuronal loss was not significant in VD, while the mixed cases with both vascular and Alzheimer-type pathology exhibited pronounced changes of neuronal density. Amyloid deposition occurred almost exclusively in AD and VA, as a rule, being of moderate degree, except for two presenile AD cases where it was marked. NFT were significantly increased in all nuclei in AD and in the VA cases, while they only occasionally appeared beyond age 55 in HD, DP and CO. The four HD cases showed in the NCS and NRD neuronal loss as severe as in AD. This neuronal loss implicates impairment of serotoninergic and noradrenergic neuromodulation as one basic mechanism promoting dementia in AD, VA and perhaps in HD.     

Brain 3,4-dihydroxyphenylethyleneglycol levels are dependent on central noradrenergic neuron activity

The effect of manipulations aimed to alter brain noradrenergic neuron activity on the levels of free and conjugated DOPEG in discrete brain areas was studied in the rat. Electrical stimulation of the medial forebrain bundle for 10–20 min produced a frequency dependent elevation of free and conjugated DOPEG concentrations in the anterior cerebral cortex and in the hippocampus. In contrast, acute interruption of noradrenergic nerve impulse flow by application of tetrodotoxin (50–100 ng) into the medial forebrain bundle markedly diminished cortical and hippocampal DOPEG levels at 0.5–2 h post-injection. Cortical conjugated and free DOPEG levels were also reduced (by 80–96%) 2–3 weeks after bilateral electrolytic lesion of the locus coeruleus, 6-hydroxydopamine-induced lesion of the ascending noradrenergic pathways or noradrenergic denervation by the neurotoxic agent DSP4. Finally, the α-adrenoceptor blocking agents yohimbine (1–10 mg/kg, ip) and RX 781094 (3–10 mg/kg, ip) increased whereas the α-adrenergic agonist clonidine (0.01–1 mg/kg, sc) decreased DOPEG levels in the cerebral cortex, hypothalamus and septal areas. These data indicate that free and conjugated DOPEG formation is dependent on, and may serve as an index of, central noradrenergic neuron activity.     

LOCALIZATION OF GABAERGIC, CHOLINERGIC AND AMINERGIC STRUCTURES IN THE MESOLIMBIC SYSTEM

Abstract— The localization of cholinergic, GABAergic and aminergic structures in the 'mesolimbic' system has been discussed from studies on the topographical distribution of choline acetyltransferase, glutamate decarboxylase and aromatic amino acid decarboxylase in normal rat brain and in brains hemitransected at the level of globus pallidus. The structures analysed included nucleus accumbens, olfactory tubercle, septum, medial forebrain bundle, striatum, substantia nigra, ventral tegmental area and nucleus interpeduncularis.
Choline acetyltranferase was highly concentrated in the nucleus interpeduncularis, but it did also exhibit considerable activity in the nucleus accumbens, the olfactory tubercle and the striatum. The activities did not change after hemitransection. Aromatic amino acid decarboxylase was highly concentrated in the ventral tegmental area, but high activities were also found in the striatum, the nucleus accumbens, the olfactory tubercle and the pars compacta of the substantia nigra. The activity decreased in all areas rostral to the hemitransection. Glutamate decarboxylase was highly concentrated in the dopamine innervated regions, moreso in the limbic structures than in the striatum. Much higher activity was found in the substantia nigra than in the ventral tegmental area. After hemitransection the activity in the substantia nigra was decreased whereas in the ventral tegmental area it was unchanged. Our results thus suggest that dopaminergic cells in the ventral tegmental area do not receive GABAergic fibres from the terminal regions of the ascending dopaminergic fibres. In addition, we found a very high concentration of glutamate decarboxylase in a region traversed by the rostral medial forebrain bundle. Here the activity was mainly confined to the paniculate fraction, probably the synaptosomes. This fraction also displayed a very active high affinity uptake of y-aminobutyric acid.     

A neurotoxic dose of methamphetamine induces gene expression of Homer 1a, but not Homer 1b or 1c, in the striatum and nucleus accumbens

Homer proteins, which regulate the signaling pathway of metabotropic glutamate receptors, may contribute to the glutamatergic modulation of dopamine neurons in the basal ganglia. This study examined whether the induction of Homer 1 genes is or not associated with the methamphetamine-induced dopaminergic neurotoxicity in the discrete brain regions of rats. Basal levels of Homer 1a and 1c mRNAs in the forebrain regions were higher than those in the substantia nigra, whereas Homer 1b mRNA levels were higher in the substantia nigra than those in the forebrain regions examined. A neurotoxic dose (40 mg/kg, i.p.) of methamphetamine increased the mRNA and protein levels of Homer 1a in the striatum and nucleus accumbens, but not in the medial prefrontal cortex or the substantia nigra. Both Homer 1b and 1c mRNAs were not affected in any brain regions examined. These results suggest that the induction of Homer 1a gene may be involved at least in part in the methamphetamine-induced dopaminergic neurotoxicity, possibly through the glutamate-dopaminergic interaction.     

Distribution of somatostatinlike immunoreactivity in the brain of the little brown bat (Myotis lucifugus)

The distribution of somatostatinlike immunoreactive (SLI) perikarya, axons, and terminals was mapped in subcortical areas of the brain of the little brown bat, Myotis lucifugus, using light microscopic immunocytochemistry. A preponderance of immunoreactivity was localized in reticular, limbic, and hypothalamic areas including: 1) in the forebrain: the bed nucleus of the stria terminalis; lateral preoptic, dorsal, anterior, lateral and posterior hypothalamic areas; amygdaloid, periventricular, arcuate, supraoptic, suprachiasmatic, ventromedial, dorsomedial, paraventricular, lateral and medial mammillary, and lateral septal nuclei; the nucleus of the diagonal band of Broca and nucleus accumbens septi; 2) in the midbrain: the periaqueductal gray, interpeduncular, dorsal and ventral tegmental, pretectal, and Edinger-Westphal nuclei; and 3) in the hindbrain: the superior central and parabrachial nuclei, nucleus incertus, locus coeruleus, and nucleus reticularis gigantocellularis. Other areas containing SLI included the striatum (caudate nucleus and putamen), zona incerta, infundibulum, supramammillary and premammillary nuclei, medial and dorsal lateral geniculate nuclei, entopeduncular nucleus, lateral habenular nucleus, central medial thalamic nucleus, central tegmental field, linear and dorsal raphe nuclei, nucleus of Darkschewitsch, superior and inferior colliculi, nucleus ruber, substantia nigra, mesencephalic nucleus of V, inferior olivary nucleus, inferior central nucleus, nucleus prepositus, and deep cerebellar nuclei. While these results were similar in some respects to those previously reported in rodents, they also provided interesting contrasts.