GABAergic and cholinergic basal forebrain and preoptic-anterior hypothalamic projections to the mediodorsal nucleus of the thalamus in the cat

The present study examined projections of GABAergic and cholinergic neurons from the basal forebrain and preoptic-anterior hypothalamus to the "intermediate" part of the mediodorsal nucleus of the thalamus. Retrograde transport from this region of the mediodorsal nucleus was investigated using horseradish peroxidase-conjugated wheatgerm agglutinin in combination with peroxidase-antiperoxidase immunohistochemical staining for glutamic acid decarboxylase and choline acetyltransferase. A relatively large number of retrogradely-labelled glutamic acid decarboxylase-positive neurons are located in the basal forebrain, amounting to more than 7% of the total population of glutamic acid decarboxylase-positive cells in this region. Moreover, retrogradely-labelled choline acetyltransferase-positive cells are interspersed among glutamic acid decarboxylase-positive neurons, accounting for about 6% of the total choline acetyltransferase-positive cell population in the basal forebrain. The glutamic acid decarboxylase-positive and choline acetyltransferase-positive retrogradely-labelled neurons are distributed throughout several regions of the basal forebrain, including the medial septum, the diagonal band of Broca, the magnocellular preoptic nucleus, the substantia innominata pars anterior, the substantia innominata pars posterior, and the globus pallidus where only a few retrogradely-labelled neurons were seen. The choline acetyltransferase-positive mediodorsal-projecting neurons are morphologically different from the choline acetyltransferase-positive neurons in the basal forebrain, suggesting that those projecting to the mediodorsal nucleus are a small proportion of the cholinergic neuronal population in the basal forebrain. In the preoptic-anterior hypothalamus, many retrogradely-labelled glutamic acid decarboxylase-positive cells were found, amounting to more than 7% of the total population of glutamic acid decarboxylase-positive cells in this region. These retrogradely-labelled glutamic acid decarboxylase-positive neurons are distributed throughout the preoptic-anterior hypothalamus in a continuous line with those in the basal forebrain, including the lateral preoptic area, the medial preoptic area, the bed nucleus of the stria terminalis, and the anterior and dorsal hypothalamic areas. The highest percentage of mediodorsal-projecting GABAergic neurons is in the anterior lateral hypothalamus where more than 25% of the total population of glutamic acid decarboxylase-positive cells project to the mediodorsal nucleus of the thalamus. Overall, of the large population of retrogradely-labelled neurons in the basal forebrain and preoptic-anterior hypothalamus, a significant proportion are glutamic acid decarboxylase-positive neurons (> 60% in the basal forebrain and > 30% in the preoptic-anterior hypothalamus), while the choline acetyltransferase-positive neurons amount to a smaller percentage of the neurons projecting to the mediodorsal nucleus (< 13% in the basal forebrain and < 2% in the preoptic-anterior hypothalamus). These results provide anatomical evidence of direct GABAergic projections from the basal forebrain and preoptic-anterior hypothalamic regions to the "intermediate" part of the mediodorsal nucleus in the cat. This GABAergic projection field could be the direct pathway by which the basal forebrain directly modulates thalamic excitability and may also be involved in mechanisms modulating electroencephalographic synchronization and sleep through the "intermediate" mediodorsal nucleus.     

Acidic fibroblast growth factor mRNA is expressed by basal forebrain and striatal cholinergic neurons

Evidence for the importance of the basal forebrain cholinergic system in the maintenance of cognitive function has stimulated efforts to identify trophic mechanisms that protect this cell population from atrophy and dysfunction associated with aging and disease. Acidic fibroblast growth factor (aFGF) has been reported to support cholinergic neuronal survival and has been localized in basal forebrain with the use of immunohistochemical techniques. Although these data indicate that aFGF is present in regions containing cholinergic cell bodies, the actual site of synthesis of this factor has yet to be determined. In the present study, in situ hybridization techniques were used to evaluate the distribution and possible colocalization of mRNAs for aFGF and the cholinergic neuron marker choline acetyltransferase (ChAT) in basal forebrain and striatum. In single-labeling preparations, aFGF mRNA-containing neurons were found to be codistributed with ChAT mRNA+ cells throughout all fields of basal forebrain, including the medial septum/diagonal band complex and striatum. By using a double-labeling (colormetric and isotopic) technique, high levels of colocalization (over 85%) of aFGF and ChAT mRNAs were observed in the medial septum, the diagonal bands of Broca, the magnocellular preoptic area, and the nucleus basalis of Meynert. The degree of colocalization was lower in the striatum, with 64% of the cholinergic cells in the caudate and 33% in the ventral striatum and olfactory tubercle labeled by the aFGF cRNA. These data demonstrate substantial regionally specific patterns of colocalization and support the hypothesis that, via an autocrine mechanism, aFGF provides local trophic support for cholinergic neurons in the basal forebrain and the striatum.     

Region-specific regulation of glutamic acid decarboxylase (GAD) mRNA expression in central stress circuits

Neurocircuit inhibition of hypothalamic paraventricular nucleus (PVN) neurons controlling hypothalamo-pituitary-adrenocortical (HPA) activity prominently involves GABAergic cell groups of the hypothalamus and basal forebrain. In the present study, stress responsiveness of GABAergic regions implicated in HPA inhibition was assessed by in situ hybridization, using probes recognizing the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65 and GAD67 isoforms). Acute restraint preferentially increased GAD67 mRNA expression in several stress-relevant brain regions, including the arcuate nucleus, dorsomedial hypothalamic nucleus, medial preoptic area, bed nucleus of the stria terminalis (BST) and hippocampus (CA1 and dentate gyrus). In all cases GAD67 mRNA peaked at 1 hr after stress and returned to unstimulated levels by 2 hr. GAD65 mRNA upregulation was only observed in the BST and dentate gyrus. In contrast, chronic intermittent stress increased GAD65 mRNA in the anterior hypothalamic area, dorsomedial nucleus, medial preoptic area, suprachiasmatic nucleus, anterior BST, perifornical nucleus, and periparaventricular nucleus region. GAD67 mRNA increases were only observed in the medial preoptic area, anterior BST, and hippocampus. Acute and chronic stress did not affect GAD65 or GAD67 mRNA expression in the caudate nucleus, reticular thalamus, or parietal cortex. Overall, the results indicate preferential upregulation of GAD in central circuitry responsible for direct (hypothalamus, BST) or multisynaptic (hippocampus) control of HPA activity. The distinct patterns of GAD65 and GAD67 by acute versus chronic stress suggest stimulus duration-dependent control of GAD biosynthesis. Chronic stress-induced increases in GAD65 mRNA expression predict enhanced availability of GAD65 apoenzyme after prolonged stimulation, whereas acute stress-specific GAD67 upregulation is consistent with de novo synthesis of active enzyme by discrete stressful stimuli.     

NGF mRNA is expressed by GABAergic but not cholinergic neurons in rat basal forebrain

Nerve growth factor (NGF) supports the survival and biosynthetic activities of basal forebrain cholinergic neurons and is expressed by neurons within lateral aspects of this system including the horizontal limb of the diagonal bands and magnocellular preoptic areas. In the present study, colormetric and isotopic in situ hybridization techniques were combined to identify the neurotransmitter phenotype of the NGF-producing cells in these two areas. Adult rat forebrain tissue was processed for the colocalization of mRNA for NGF with mRNA for either choline acetyltransferase, a cholinergic cell marker, or glutamic acid decarboxylase, a GABAergic cell marker. In both regions, many neurons were single-labeled for choline acetyltransferase mRNA, but cells containing both choline acetyltransferase and NGF mRNA were not detected. In these fields, virtually all NGF mRNA-positive neurons contained glutamic acid decarboxylase mRNA. The double-labeled cells comprised a subpopulation of GABAergic neurons; numerous cells labeled with glutamic acid decarboxylase cRNA alone were codistributed with the double-labeled neurons. These data demonstrate that in basal forebrain GABAergic neurons are the principal source of locally produced NGF.     

Castration decreases single cell levels of mRNA encoding glutamic acid decarboxylase in the diagonal band of broca and the sexually dimorphic nucleus of the preoptic area

Using quantitative in situ hybridization histochemistry (ISHH), we determined the effect of castration on single cell levels of glutamic acid decarboxylase (GAD) mRNA in discrete hypothalamic regions of the male rat brain associated with the control of gonadotropin secretion. A 48-base oligodeoxynucleotide probe was used to detect with equal affinity the two isoforms of GAD message, GAD65 and GAD67. GAD message also was quantitated in a number of selected areas of the brain to contrast GAD gene expression amongst several populations of GABAergic neurons. Comparison of 11 brain regions demonstrated a 9.3-fold range in the quantity of single cell GAD mRNA with levels being highest in the amygdala and the diagonal band of Broca, moderate in the piriform cortex, caudate nucleus, substantia innominata, globus pallidus, cingulate cortex and medial septal nucleus, and lowest in the lateral septal nucleus and the medial preoptic nucleus (MPN). Castration markedly reduced single cell GAD mRNA levels in the DBB and the MPN, two discrete hypothalamic structures known to contain dendritic fields, cell bodies, and axons of GnRH neurons projecting to the median eminence. A striking finding was a dense core of steroid-sensitive GABAergic neurons within the MPN comprising the sexually dimorphic nucleus of the preoptic area (SDN-POA). Similar to the MPN as a whole, the amount of GAD mRNA expressed by cells in the SDN-POA of sham operated control rats was greater than in castrated animals. GAD mRNA levels were inversely related to serum LH titers, suggesting a role for these neurons in the mechanism controlling gonadal steroid negative feedback on LH secretion. This report provides the basis for future work to determine if GAD65, GAD67 or whether both isoforms are affected by gonadal steroid input.     

An alternate pathway for visual signal integration into the hypothalamo-pituitary axis: retinorecipient intergeniculate neurons project to various regions of the hypothalamus and innervate neuroendocrine cells including those producing dopamine

Using tract tracing and immunocytochemistry, this study explored the connectivity between lateral geniculate efferents and neurons of the hypothalamus, including those producing dopamine, that have direct access to fenestrated capillaries. It was also determined whether the intergeniculate neurons that give rise to hypothalamic projections are targeted by retinal axons. Within the hypothalamus, Phaseolus vulgaris leucoagglutinin-labeled, lateral geniculate efferents were observed in the suprachiasmatic nucleus, subparaventricular area, periventricular nuclei, medial preoptic areas, and between the arcuate and ventromedial nuclei. In these sites, intergeniculate efferents contacted populations of neurons that were retrogradely labeled from fenestrated capillaries by the intraperitoneal injection of fluorogold. Hypothalamic dopamine neurons, a population of which was neuroendocrine, were also synaptic targets of lateral geniculate efferents. After injection of the retrograde tracer fluorogold into these hypothalamic projection sites in parallel with bilateral enucleation, retrogradely labeled perikarya were restricted to the intergeniculate leaflet. All of the labeled perikarya contained infolded nuclei, and their distal dendrites were frequently found to be contacted by degenerated, retinal fibers. This study provides morphological evidence for a signaling pathway from the retina through the intergeniculate leaflet to hypothalamic cells that participate in neuroendocrine regulations. These observations raise the possibility that visual signals independent of the circadian clock may also influence the hypothalamo-pituitary axis. In light of the overlapping distribution of intergeniculate and suprachiasmatic efferents in the hypothalamus and their similar relationship with neuroendocrine cells, it is suggested that integration of circadian and visual signals can occur outside of the suprachiasmatic nucleus to regulate endocrine rhythms.     

Fluctuations in relative levels of choline acetyltransferase mRNA in different regions of the rat basal forebrain across the estrous cycle: effects of estrogen and progesterone

Quantitative in situ hybridization techniques were used to compare relative cellular levels of choline acetyltransferase (ChAT) mRNA in different regions of the female rat basal forebrain at different stages of the estrous cycle and at different time points after the administration of physiological levels of estrogen and progesterone. Significant fluctuations in relative levels of ChAT mRNA were detected during the course of the estrous cycle. In the medial septum (MS) and striatum, the highest levels of ChAT mRNA were detected on diestrus 1. Fluctuations in the nucleus basalis magnocellularis (NBM) were highly variable, with the highest levels detected on diestrus 2. In ovariectomized animals, significant increases in ChAT mRNA were detected in the MS, NBM, and striatum within 1-3 d after a single administration of estradiol. In addition, the effects of estradiol on ChAT mRNA expression in the NBM and striatum were significantly enhanced by the subsequent administration of progesterone. The magnitude and timing of the effects of steroid replacement were consistent with the magnitude and time course of the fluctuations detected during the course of the estrous cycle. These data demonstrate that estrogen and progesterone can increase basal forebrain levels of ChAT mRNA significantly in specific regions of the rat basal forebrain, that the magnitude and time course of the effects vary between different subpopulations of cholinergic neurons, and that the effects are associated with changes in the functioning of specific basal forebrain cholinergic neurons across the estrous cycle.     

Evidence for a GABAergic projection from the central nucleus of the amygdala to the brainstem of the macaque monkey: a combined retrograde tracing and in situ hybridization study

The central nucleus of the amygdala is interconnected with a variety of visceral and autonomic nuclei of the brainstem. These include the parabrachial nucleus, the nucleus of the solitary tract, the nucleus ambiguus and the dorsal motor nucleus of the vagus. Despite repeated attempts, neurochemical characterization of the major subcortical connections of the central nucleus has not yet been accomplished. Based on earlier immunohistochemical and in situ hybridization evidence indicating the presence of numerous GABAergic neurons in the macaque monkey central nucleus, we predicted that a sizeable portion of the descending projections may be GABAergic. We tested this hypothesis using a novel double labelling method with gold conjugated WGA-apoHRP as a retrograde tracer and in situ hybridization for detecting the mRNA that encodes the enzyme glutamic acid decarboxylase (GAD67) as a marker for GABAergic cells. Following WGA-apoHRP-gold injections into the brainstem, a large number of retrogradely labelled cells was observed in the medial and lateral divisions of the central nucleus. Of the retrogradely labelled cells observed in the medial division of the central nucleus, approximately half were double-labelled for GAD67 mRNA; about 30% double labelling was observed in the lateral division. These data support the view that a sizeable component of the central nucleus projection to the brainstem is GABAergic.     

Basal ganglia organization in amphibians: catecholaminergic innervation of the striatum and the nucleus accumbens

The aim of the present study was to determine the origin of the catecholaminergic inputs to the telencephalic basal ganglia of amphibians. For that purpose, retrograde tracing techniques were combined with tyrosine hydroxylase immunohistochemistry in the anurans Xenopus laevis and Rana perezi and the urodele Pleurodeles waltl. In all three species studied, a topographically organized dopaminergic projection was identified arising from the posterior tubercle/mesencephalic tegmentum and terminating in the striatum and the nucleus accumbens. Although essentially similar, the organization of the mesolimbic and mesostriatal connections in anurans seems to be more elaborate than in urodeles. The present study has also revealed the existence of a noradrenergic projection to the basal forebrain, which has its origin in the locus coeruleus. Additional catecholaminergic afferents to the striatum and the nucleus accumbens arise from the nucleus of the solitary tract, where catecholaminergic neurons appear to give rise to the bulk of the projections to the basal forebrain. In other regions, such as the olfactory bulb, the anterior preoptic area, the suprachiasmatic nucleus, and the thalamus, retrogradely labeled neurons (after basal forebrain tracer-applications) and catecholaminergic cells were intermingled, but none of these centers contained double-labeled cell bodies. It is concluded that the origin of the catecholaminergic innervation of the striatum and the nucleus accumbens in amphibians is largely comparable to that in amniotes. The present study, therefore, strongly supports the existence of a common pattern in the organization of the catecholaminergic inputs to the basal forebrain among tetrapod vertebrates.     

Increased ipsilateral expression of Fos following lateral hypothalamic self-stimulation

Immunohistochemical labeling of Fos protein was used to visualize neurons activated by rewarding stimulation of the lateral hypothalamic level of the medial forebrain bundle (MFB). Following training and stabilization of performance, seven rats were allowed to self-stimulate for 1 h prior to anesthesia and perfusion. Brains were then processed for immunohistochemistry. Two control subjects were trained and tested in an identical manner except that the stimulator was disconnected during the final 1 h test. Among the structures showing a greater density of labeled neurons on the stimulated side of the brains of the experimental subjects were the septum, lateral preoptic area (LPO), medial preoptic area, bed nucleus of the stria terminalis, substantia innominata (SI), and the lateral hypothalamus (LH). Several of these structures, the LPO, SI, and LH, have been implicated in MFB self-stimulation by the results of psychophysical, electrophysiological, and lesion studies.     

Cellular localizations of AMPA glutamate receptors within the basal forebrain magnocellular complex of rat and monkey

The cellular distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors within the rodent and nonhuman primate basal forebrain magnocellular complex (BFMC) were demonstrated immunocytochemically using anti-peptide antibodies that recognize glutamate receptor (GluR) subunit proteins (i.e., GluR1, GluR4, and a conserved region of GluR2, GluR3, and GluR4c). In both species, many large GluR1-positive neuronal perikarya and aspiny dendrites are present within the medial septal nucleus, the nucleus of the diagonal band of Broca, and the nucleus basalis of Meynert. In this population of neurons in rat and monkey, GluR2/3/4c and GluR4 immunoreactivities are less abundant than GluR1 immunoreactivity. In rat, GluR1 does not colocalize with ChAT, but, within many neurons, GluR1 does colocalize with GABA, glutamic acid decarboxylase (GAD), and parvalbumin immunoreactivities. GluR1- and GABA/GAD-positive neurons intermingle extensively with ChAT-positive neurons. In monkey, however, most GluR1-immunoreactive neurons express ChAT and calbindin-D28 immunoreactivities. The results reveal that noncholinergic GABAergic neurons, within the BFMC of rat, express AMPA receptors, whereas cholinergic neurons in the BFMC of monkey express AMPA receptors. Thus, the cellular localizations of the AMPA subtype of GluR are different within the BFMC of rat and monkey, suggesting that excitatory synaptic regulation of distinct subsets of BFMC neurons may differ among species. We conclude that, in the rodent, BFMC GABAergic neurons receive glutamatergic inputs, whereas cholinergic neurons either do not receive glutamatergic synapses or utilize GluR subtypes other than AMPA receptors. In contrast, in primate, basal forebrain cholinergic neurons are innervated directly by glutamatergic afferents and utilize AMPA receptors.     

EYA4, a novel vertebrate gene related to Drosophila eyes absent

NMDA receptors are composed of proteins from two families: NMDAR1 and NMDAR2. We used quantitative double-label in situ hybridization to examine in rat brain the expression of NMDAR1, NMDAR2A, NMDAR2B, and NMDAR2C mRNA in six neurochemically defined populations of striatal neurons: preproenkephalin (ENK) and preprotachykinin (SP) expressing projection neurons, and somatostatin (SOM), glutamic acid decarboxylase 67 (GAD67), parvalbumin (PARV), and choline acetyltransferase (ChAT) expressing interneurons. NMDAR1 was expressed by all striatal neurons: strongly in ENK, SP, PARV and ChAT neurons, and less intensely in SOM and GAD67 positive cells. NMDAR2A mRNA was present at moderate levels in all striatal neurons except those containing ChAT. Labeling for NMDAR2B was strong in projection neurons and ChAT interneurons, and only moderate in SOM, GAD67 and PARV interneurons. NMDAR2C was scarce in striatal neurons, but a low level signal was detected in GAD67 positive cells. NMDAR2C expression was also observed in small cells not labeled by any of the markers, most likely glia. These data suggest that all striatal neurons have NMDA receptors, but different populations have different subunit compositions which may affect function as well as selective vulnerability.     

Afferent and efferent connections of the diencephalic prepacemaker nucleus in the weakly electric fish, Eigenmannia virescens: interactions between the electromotor system and the neuroendocrine axis

The organization of the ventral nucleus of the ventral telencephalon (Vv) was examined in the weakly electric fish, Eigenmannia virescens. This nucleus, which is considered the teleost homologue to the basal forebrain nuclei of other vertebrates, was subdivided into dorsal and ventral subdivisions, based upon cytoarchitectonic, immunohistochemical, and connectional criteria. Afferent projections were observed from the medial olfactory bulb as well as the terminal nerve ganglion. Telencephalic afferents to the Vv were very restricted, consisting of the supracommissural and the dorsal intermediate nuclei of the ventral telencephalon, the nucleus taenia, and the medial region of the posterior nucleus of the dorsal telencephalon. However, the major afferents to the Vv were diencephalic, particularly those originating from the rostral preoptic area and other hypothalamic nuclei. Additional afferents included the posterior tubercular nucleus, the locus coeruleus, the medial perilemniscal nucleus, and the periventricular nucleus of the posterior tuberculum. Relatively weak projections were observed from the ventral thalamus and the dorsal posterior thalamic nucleus. As described previously, the diencephalic complex of the central posterior thalamic nucleus/prepacemaker nucleus (CP/PPn), which also has cells that innervate the pacemaker circuitry controlling the production of an electric organ discharge, projects to the Vv. Terminal fields of the Vv were observed to be coextensive with afferent cell groups in the preoptic area, lateral and caudal hypothalamic nuclei, and thalamus. An additional efferent target of the Vv was the pretectal nucleus electrosensorius. That many cell groups that are connected with the Vv are also connected with the CP/PPn, particularly the preoptic and hypothalamic nuclei, suggests that the electrocommunicatory system is intimately linked with basal forebrain limbic pathways.     

Parasolitary nucleus: a source of GABAergic vestibular information to the inferior olive of rat and rabbit

At least two subnuclei of the inferior olive, the beta-nucleus, and the dorsomedial cell column (dmcc), contain vestibularly responsive neurons that receive a dense descending projection that uses gamma-aminobutyric acid (GABA) as the transmitter. In contrast to the GABAergic innervation of other olivary subnuclei, the terminal boutons that terminate on neurons in the beta-nucleus and the dorsomedial cell column remain intact after cerebellectomy, ruling out both the cerebellum and the cerebellar nuclei as afferent sources. By using both immunohistochemical as well as orthograde and retrograde tracer methods, we have identified the source of the GABAergic pathway to the beta-nucleus and dmcc in both rat and rabbit. Under physiologic recording of single olivary neurons to guide electrode placement, we injected the bidirectional tracer, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the beta-nucleus and dmcc of the inferior olive. These injections retrogradely labeled neurons in the parasolitary nucleus (Psol) near the vestibular complex. Psol neurons were identified as GABAergic with an antibody to glutamic acid decarboxylase (GAD). In the rat, Psol neurons are small (5-7 microm in diameter) and number approximately 1,800. In the rabbit, they are slightly larger (6-9 microm in diameter) and number approximately 2,200. WGA-HRP injections in conjunction with GAD immunohistochemistry double labeled a high percentage of neurons in both the rat and rabbit Psol. Injection of the orthograde tracer Phaseolus vulgaris-leucoagglutinin into the area of the Psol revealed a projection from this region to both the beta-nucleus and dmcc. Subtotal electrolytic lesions of this division of the Psol caused a substantial reduction in GAD-positive synaptic terminals in both the ipsilateral beta-nucleus and dmcc. The location of these GABAergic neurons, bordering both the nucleus solitarius and caudal vestibular complex, emphasizes the importance of the Psol in the processing of both vestibular and autonomic information pertinent to postural control.     

The basal forebrain. Neuroanato-physiology and neurologic applications

The Basal Forebrain region comprises the heterogeneous set of telencephalic structures on the medial and ventral aspects of the cerebral hemisphere. Basal Forebrain structures include the septal areas, olfactory tubercule, substantia innominata, and parts of the amygdala. The basal forebrain region is the site of a system of cholinergic neurons projecting to the entire cortical surface. The Basal Forebrain is a site of convergence of sleep and thermoregulatory functions. Portions of the basal forebrain play prominent roles in the control of the body temperature. The medial preoptic/anterior hypothalamic areas have both thermosensiting and thermointegrating functions. Cholinergic neurons intrinsic to the Basal Forebrain play a fundamental role in sleep onset and maintenance. Neurons of the nucleus basalis of Meynert undergo a profound degeneration in patients with Alzheimer's disease and provide a pathological substrate of the cholinergic deficiency in their brain. Behavior changes and amnesic syndrome may complicate surgery for ruptured anterior communicating artery aneurysms. Disruption of the blood supply through the perforating branches of the anterior communicating artery may probably cause the amnesic syndrome.     

Recognition and management of childhood cardiac arrhythmias

Axonal connections between the amygdala and the hypothalamic paraventricular nucleus were examined by combined anterograde-retrograde tract tracing. Iontophoretic injections of the retrograde tracer Fluorogold were placed in the paraventricular nucleus, and the anterograde tracer PHA-L in the ipsilateral central or medial amygdaloid nuclei. Single and double-label immunohistochemistry were used to detect tracers. Single label anterograde and retrograde tracing suggest limited evidence for direct connections between the central or medial amygdala and the paraventricular nucleus. In general, scattered PHA-L-positive terminals were seen in autonomic subdivisions of the paraventricular nucleus (lateral parvocellular, dorsal parvocellular and ventral medial parvocellular subnuclei) following central or medial amygdaloid nucleus injection. Double-label studies indicate that central and medial amygdaloid nucleus efferents contact paraventricular nucleus-projecting cells in several forebrain nuclei. In the case of central nucleus injections, PHA-L positive fibers occasionally contacted Fluorogold-labeled neurons in the anteromedial, ventromedial and preoptic subnuclei of the bed nucleus of the stria terminalis. Overall, such contacts were quite rare, and did not occur in the bed nucleus of the stria terminalis regions showing greatest innervation by the central amygdaloid nucleus. In contrast, medial amygdala injections resulted in a significantly greater overlap of PHA-L labeling and Fluorogold-labeled neurons, with axosomatic appositions observed in medial divisions of the bed nucleus of the stria terminalis, anterior hypothalamic area and preoptic area. The results provide anatomical evidence that a substantial proportion of amygdaloid connections with hypophysiotrophic paraventricular nucleus neurons are likely multisynaptic, relaying in different subregions of the bed nucleus of the stria terminalis and hypothalamus.     

Heterogeneity and selectivity of the degeneration of cholinergic neurons in the basal forebrain of patients with Alzheimer's disease

Cholinergic neurons were studied by immunohistochemistry, with an antiserum against choline acetyltransferase (ChAT), in the basal forebrain (Ch1 to Ch4) of four patients with Alzheimer's disease (AD) and four control subjects. ChAT-positive cell bodies were mapped and counted in Ch1 (medial septal nucleus), Ch2 (vertical nucleus of the diagonal band), Ch3 (horizontal nucleus of the diagonal band) and Ch4 (nucleus basalis of Meynert). Compared to controls, the number of cholinergic neurons in AD patients was reduced by 50% on average. The interindividual variations in cholinergic cell loss were high, neuronal loss ranging from moderate (27%) to severe (63%). Despite the small number of brains studied, a significant correlation was found between the cholinergic cell loss and the degree of intellectual impairment. To determine the selectivity of cholinergic neuronal loss in the basal forebrain of AD patients, NPY-immunoreactive neurons were also investigated. The number of NPY-positive cell bodies was the same in controls and AD patients. The results (1) confirm cholinergic neuron degeneration in the basal forebrain in AD and the relative sparing of these neurons in some patients, (2) indicate that degeneration of cholinergic neurons in the basal forebrain contributes to intellectual decline, and (3) show that, in AD, such cholinergic cell loss is selective, since NPY-positive neurons are preserved in the basal forebrain.     

Origin, course, and laterality of spinocerebellar axons in the North American opossum, Didelphis virginiana

Inhibition of neurons containing gamma-aminobutyric acid (GABA) may underlie some of the excitatory effects of opioids in the central nervous system (CNS). In the present study, we examined the relationship of the cloned mu- and delta-opioid receptors (MOR1 and DOR1, respectively) to GABAergic neurons in brain and spinal cord. This was done by combining immunofluorescent staining for MOR1 or DOR1 with that for GABA or glutamic acid decarboxylase (GAD); fluorescent retrograde tract-tracing was used in some cases to identify neurons with particular projections. In rats, cells double labeled for GABA and MOR1 were observed in layers II-VI of the parietal cortex and in layers II-IV of the piriform cortex. In the hippocampus, double labeling was observed in the dentate gyrus and in regions CA1 and CA3. Double labeling was very prominent in the striatum and in the reticular nucleus of the thalamus; it was also observed in other portions of the diencephalon. However, double labeling for GABA and MOR1 was never observed in the cerebellar cortex. Cells double labeled for GABA and MOR1 were common in the periaqueductal gray (PAG) and the medial rostral ventral medulla (RVM) of both rats and monkeys, suggesting that involvement of GABAergic neurons with supraspinal opioid antinociception may extend to primates. In the RVM of rats, many of those double-labeled neurons were retrogradely labeled from the dorsal spinal cord. In contrast, double-labeled neurons in the PAG were almost never retrogradely labeled from the RVM. No unequivocal examples of double labeling for DOR1 and GAD were found in any region of the CNS that we examined in either rats or monkeys. However, GABAergic neurons were often apposed by DOR1 immunoreactive varicosities. Our findings suggest that activation of mu-opioid receptors directly modulates the activity of GABAergic neurons throughout the CNS, including neurons involved in the supraspinal component of opioid analgesia. In contrast, delta-opioid receptors appear to be positioned to modulate the activity of GABAergic neurons indirectly.     

A topographically organized gamma-aminobutyric acid projection from the ventral pallidum to the nucleus accumbens in the rat

Anatomical and electrophysiological studies have indicated that a reciprocal projection from the ventral pallidum back to the nucleus accumbens exists and has functional relevance. In this study, the topographical projection from the ventral pallidum to the nucleus accumbens was examined by using retrograde tracing with fluoro-gold iontophoresed in subcompartments of the nucleus accumbens in rats combined with either in situ hybridization for glutamic acid decarboxylase and preproenkephalin mRNA or substance P immunoreactivity. Deposits made into the medial nucleus accumbens preferentially labeled neurons in the medial ventral pallidum, while deposits into the dorsolateral nucleus accumbens, at or lateral to the anterior commissure, labeled primarily cells in the dorsal and lateral ventral pallidum. A mediolateral to rostrocaudal topography was also observed, with the medial deposits preferentially labeling cells in rostral ventral pallidum and the lateral deposits resulting in retrogradely labeled cells in the ventral pallidum below the crossing of the posterior anterior commissure (subcommissural) as well as below the globus pallidus (sublenticular). The majority of cells retrogradely labeled with fluoro-gold were double-labeled for glutamic acid decarboxylase mRNA. In contrast, very few retrogradely labeled neurons in the ventral pallidum were double labeled for mRNA for preproenkephalin. These data demonstrate a topographically organized projection from the ventral pallidum to the nucleus accumbens that is primarily gamma-aminobutyric acid (GABA)-ergic and reciprocal to the GABAergic projection from the nucleus accumbens to the ventral pallidum.