The effects of androgen on the density of spine synapses on pyramidal neurons in the CA1 area of the hippocampus were studied in male rats. Gonadectomy (GDNX) had no significant effect on the number of CA1 pyramidal cells but reduced CA1 spine synapse density by almost 50% (to 0.468 +/- 0.018 spine synapses/microm(3)) compared with sham-operated controls (0.917 +/- 0.06 spine synapses/microm(3)). Treatment of GDNX rats with testosterone propionate (500 microg/d, s.c., 2 d) increased spine synapse density to levels (1.01 +/- 0.026 spine synapses/microm(3)) comparable with intact males. A similar increase in synapse density (1.013 +/- 0.05 spine synapses/microm(3)) was observed in GDNX animals after treatment with dihydrotestosterone (DHT) (500 microg/d, s.c., 2 d) but not after estradiol (10 microg/d, s.c., 2 d; 0.455 +/- 0.02 spine synapse/microm(3)). These data indicate that testosterone is important for maintenance of normal spine synapse density in the CA1 region of the male rat hippocampus. The comparable responses to testosterone and the non-aromatizable androgen DHT, coupled with the lack of response to estradiol, suggest that testosterone acts directly on hippocampal androgen receptors rather than indirectly via local estrogen biosynthesis.

The cholinergic innervation of the rat hippocampus proper and fascia dentata was investigated by using a monoclonal antibody against choline acetyltransferase (ChAT). At the light microscopic level, thin varicose ChAT-immunoreactive fibers were observed mainly in the vicinity of the pyramidal and granular layers where they formed a fine network around proximal dendrites of pyramidal and granule cells. In addition, many ChAT-immuno-reactive fibers were found in the hilar region and in stratum oriens, radiatum, and lacunosum-moleculare of all hippocampal sectors. Electron microscopic analysis revealed ChAT immunoreactivity in thin unmyelinated varicose axons and terminals which established synaptic contacts. Asymmetric contacts of ChAT-immunoreactive terminals were found on small spines in the dendritic layers of the hippocampus proper and in the molecular layer of the fascia dentata. Symmetric synaptic contacts were formed on the cell bodies of pyramidal and granule cells. Both symmetric and asymmetric synaptic contacts occurred on dendritic shafts. The analysis of serial thin sections, which allows identification of postsynaptic elements, suggests that pyramidal cells, granule cells, and nonpyramidal neurons of the hippocampus receive a cholinergic input.

The medial prefrontal cortex and the hippocampus serve well recognized roles in memory processing. The hippocampus projects densely to, and exerts strong excitatory actions on, the medial prefrontal cortex. Interestingly, the medial prefrontal cortex, in rats and other species, has no direct return projections to the hippocampus, and few projections to parahippocampal structures including the entorhinal cortex. It is well established that the nucleus reuniens of the midline thalamus is the major source of thalamic afferents to the hippocampus. Since the medial prefrontal cortex also distributes to nucleus reuniens, we examined medial prefrontal connections with populations of nucleus reuniens neurons projecting to hippocampus. We used a combined anterograde and retrograde tracing procedure at the light and electron microscopic levels. Specifically, we made Phaseolus vulgaris-leuccoagglutinin (PHA-L) injections into the medial prefrontal cortex and Fluorogold injections into the hippocampus (CA1/subiculum) and examined termination patterns of anterogradely PHA-L labeled fibers on retrogradely FG labeled cells of nucleus reuniens. At the light microscopic level, we showed that fibers from the medial prefrontal cortex form multiple putative synaptic contacts with dendrites of hippocampally projecting neurons throughout the extent of nucleus reuniens. At ultrastructural level, we showed that medial prefrontal cortical fibers form asymmetric contacts predominantly with dendritic shafts of hippocampally projecting reuniens cells. These findings indicate that nucleus reuniens represents a critical link between the medial prefrontal cortex and the hippocampus. We discuss the possibility that nucleus reuniens gates the flow of information between the medial prefrontal cortex and hippocampus dependent upon attentive/arousal states of the organism.

The medial septum/diagonal band (MSDB), which gives rise to the septohippocampal pathway, is a critical locus for the mnemonic effects of muscarinic drugs. Infusion of muscarinic cholinergic agonists into the MSDB enhance learning and memory processes both in young and aged rats and produce a continuous theta rhythm in the hippocampus. Intraseptal muscarinic agonists also alleviate the amnesic syndrome produced by systemic administration of muscarinic receptor antagonists. It has been presumed, but not proven, that the cellular mechanisms underlying the effects of muscarinic agonists in the MSDB involve an excitation of septohippocampal cholinergic neurons and a subsequent increase in acetylcholine (ACh) release in the hippocampus. Using a novel fluorescent labeling technique to selectively visualize live septohippocampal cholinergic neurons in rat brain slices, we have found that muscarinic agonists do not excite septohippocampal cholinergic neurons, instead they inhibit a subpopulation of cholinergic neurons. In contrast, unlabeled neurons, confirmed to be noncholinergic, septohippocampal GABA-type neurons using retrograde marking and double-labeling techniques, are profoundly excited by muscarine. Thus, the cognition-enhancing effects of muscarinic drugs in the MSDB cannot be attributed to an increase in hippocampal ACh release. Instead, disinhibitory mechanisms, caused by increased impulse flow in the septohippocampal GABAergic pathway, may underlie the cognition-enhancing effects of muscarinic agonists.