The human hippocampal formation and parahippocampal gyrus: localization of substance P-like immunoreactivity in newborn and adult post-mortem tissue.

The localization of substance P-like immunoreactive nervous structures in the human post-mortem hippocampal formation and parahippocampal gyrus was studied by the indirect immunofluorescence technique of Coons and Kaplan (1950), J. exp. Med. 91, 1-9. Specimens were obtained from brains of both newborn and adult subjects. Substance P-positive perikarya were easily detectable in these regions, being particularly abundant in the stratum oriens of the hippocampus and in the hilus of the fascia dentata. The nerve fibres and terminals were also widely and unevenly distributed. Comparison of the newborn and adult tissue revealed differences in the distribution of substance P-like immunoreactivity in ontogenetic stages. Our results, together with data available in the literature, further suggest that substance P is differently distributed in the various cortical areas of the human brain, and that the distribution of substance P may vary among species.

Mechanisms for sensory adaptation in frog vestibular organs.

The effects of external low-Ca, high-Mg solutions were tested both on frog isolated semicircular canals and on single cells isolated from these sensory organs. Our results showed that these media were able to cancel slow adaptation of the ampullar microphonic current in the whole organ and to abolish a Ca-dependent K current (IK(Ca)) in single hair cells, suggesting that IK(Ca) is involved in vestibular sensory adaptation.

NMDA receptors in frog semicircular canals.

The effects of competitive (2-amino-5-phosphonovaleric acid) and noncompetitive (Mg2+, ketamine and kynurenic acid) NMDA receptor antagonists were tested in isolated semicircular canals of the frog. All these antagonists reduced the resting firing rate recorded from the whole ampullar nerve of the posterior canal. This suggests that NMDA receptors are present at the cytoneural junctions between hair cells and afferent nerve terminals in frog vestibular organs.

Calcium channels functional roles in the frog semicircular canal.

Different types of voltage-operated calcium channels have been described in hair cells; however, no clear functional role has been assigned to them. As a first functional characterization of vestibular calcium channels, we studied the effect of several calcium channel agonists and antagonists on whole nerve firing rate in an isolated frog semicircular canal preparation. Resting activity was affected by all dihydropyridines tested and by omegaconotoxin GVIA, whereas only nimodipine was able to reduce the mechanically evoked activity. These results indicate that nimodipine-sensitive channels play a major role in afferent transmitter release, and omega-conotoxin GVIA sensitive channels regulate the afferent firing (possibly on the postsynaptic side) but with a less important role.

beta amyloid-induced disruption of ionic balance: studies on the isolated frog labyrinth.

The beta-amyloid peptide (A beta) is a key player in the pathogenesis of Alzheimer's disease. Although its mechanisms of action are not fully elucidated, a disruption of ionic homeostasis has been suggested, and A beta aggregation in fibrils seems correlated to its toxic potential. In the present work, we studied the effects of different A beta fragments on the activity of frog ampullar nerve fibers. Our results show that A beta fragments are able to reduce ampullar nerve responses, with a potency correlated to their fibrillogenic capability. This study may have clinical implications, since vestibular problems are often reported in Alzheimer patients, and provide a model for the dissection of A beta effects in a simple multicomponent system.

Caloric stimulation of ampullar receptors: a new method to produce mechanically-evoked responses in frog semicircular canals.

A microthermistor positioned close to the exposed posterior semicircular canal in isolated labyrinth preparations of the frog was used to stimulate the sensory organ. Our results indicated that, depending on the position of the heater, the induced endolymphatic convection currents may result in either excitatory or inhibitory cupular deflections and thus in a modulation of ampullar receptor resting activity. Other possible thermal-dependent mechanisms, such as a direct action of the stimulus on vestibular sensors or endolymphatic volume changes, had, in the present experimental conditions, a minor role. Caloric stimulation could therefore represent a novel method to stimulate the semicircular canals 'in situ'.

Functional organization of the peripheral efferent vestibular system in the frog.

The functional organization of the efferent vestibular system (EVS) was studied in the isolated frog labyrinth. To ascertain whether, besides the efferent branching fibres that innervate several end-organs, the EVS is also endowed with efferent non-branching axons which might control a given population of sensory units in each end-organ, the 8th nerve and one of its branchlets were electrically stimulated while recordings of the spontaneous activity arising from the different sensors were made by impaling single afferent axons in all the 8th nerve branchlets. The results demonstrated that the vast majority of the sensory units whose activity was modified by stimulating the whole 8th nerve was also affected by stimulating an 8th nerve branchlet. These findings therefore rule out the possibility that the EVS is endowed with projective fibres and strengthen the view that the EVS is a highly divergent system with collaterals arising from single parent axons that innervate several end-organs. These experiments have also shown that the percentage of sensory units which are actually controlled by the EVS varies amongst the different labyrinthine organs. It is maximal in the sacculus (ca. 90%), somewhat lower in canal organs (ca. 80%) and the utriculus (ca. 70%) and considerably lower in the lagena (ca. 50%). This EVS arrangement therefore might allow information arising from some organs to be modified more extensively than that from others.

The effect of glutamate on the frog semicircular canal.

L-glutamate (Glu) has at least two sites of action in the frog semicircular canal: the hair cell (presynaptic) and the primary afferent nerve fibres (postsynaptic). Glu's action on the hair cell results in an increased release of the natural transmitter which is responsible for a substantial increase in the frequency of firing in primary afferents. The presynaptic action of Glu is antagonized by D-alpha-aminoadipate (D alpha AA). Glu produces a long-lasting depolarization on the afferent nerve fibres which does not by itself elicit any afferent discharge of impulses when the release of the natural transmitter is prevented. Glu-induced nerve depolarization is only partially antagonized by D alpha AA. The difficulty of reconciling some of the observations made of the effects of Glu in semicircular canals with its presumed role as an afferent transmitter in this organ is discussed, but this role is not definitely rejected.

Evidence for the presence of substance P-like immunoreactivity in the human cerebellum.

Preliminary results on the localization of substance P-like immunoreactivity in the human cerebellum are presented. Cerebella from newborn and adult subjects were examined. While only sporadic positive fibres were detected in the adult tissue, the immunoreactive material appeared more abundant in the cerebella from newborn subjects. Varicose and non-varicose fibres and dot-like nerve terminals were present with different density in various regions. The paucity of immunoreactive perikarya suggests that most of the cerebellar substance P-like immunoreactive material has an extrinsic origin.

Sensory adaptation in frog vestibular organs.

Adaptation, i.e., the decrease with time in sensory units' afferent discharge to a constant stimulus, appears to be a common feature of the receptors belonging to acoustico-lateralis system: However, the mechanisms underlying this process are still a matter of debate. The present experiments demonstrate that sensory adaptation to both mechanical and electrical stimuli can be nearly suppressed after perilymphatic ouabain administration. This clearly indicates that the K+ homeostatic mechanisms [Valli et al., (1990) J. Physiol. (London) 430, 585-594] which control the K+ concentration gradient at both ends of vestibular hair cells play a predominant role in this process. The possible importance of different K+-dependent mechanisms in hair cell adaptation is discussed.

Evidence for L-glutamate release in frog vestibular organs.

The present study was devised in order to ascertain whether L-glutamate (Glu) is the neurotransmitter at the primary afferent synapse in frog vestibular organs. To this end different groups of frog isolated semicircular canals were stimulated by means of solutions slightly enriched in K+ (5 mM K(+)-rich solutions are sufficient to produce a strong, long-lasting, transmitter release from the basal pole of sensory cells) both in normal conditions and after low-Ca(2+)-high-Mg2+ impairment of the synaptic transmission. The concentration of Glu in the surrounding medium, determined by means of a bioluminescence-enzymatic method, was evaluated in two different experimental conditions: a) when the canals (5 canals placed inside little net bags) were immersed in a 5 mM K(+)-stimulating solution; b) during the superfusion of the canals (25 canals placed into a little perfusion chamber) with a 5 mM K(+)-stimulating solution. The net bag experiments demonstrated that K(+)-rich solutions can provoke an outflow of Glu from canal organs only if the crista ampullaris is present and functioning. Glu fluctuations were in fact suppressed by employing canals deprived of the ampulla or after low-Ca2(+)-high-Mg2+ synaptic blockade. The superfusion experiments demonstrated that the time course of 5 mM K(+)-induced release of Glu from the sensory organ strictly parallels the time course of 5 mM K(+)-induced EPSPs and spike discharge in afferent axons. These results strongly support the hypothesis that Glu is, or is released with, the afferent transmitter in frog inner ear sensory organs.

Streptomycin blocks the afferent synapse of the isolated semicircular canals of the frog.

This study aimed to define the acute electrophysiological effects of the perilymphatic perfusion of streptomycin in the sensory apparatus of the semicircular canals of the frog. The ampullary DC potential, the vestibular nerve multiunit discharge, the nerve DC potential and the unitary EPSP activity were recorded in isolated semicircular canals of the frog (Rana esculenta L). The results demonstrated that perilymphatic microperfusion of streptomycin (0.1, 0.3, 1 and 3 mM) reduced both resting and mechanically evoked afferent discharge, while the response of the hair cells remains unchanged. Intracellular recordings from single afferent axons showed that the reduction of the afferent discharge was mainly due to a reduction of the amplitude, but not the frequency, of the EPSPs. These results indicate that streptomycin, when applied in the fluid bathing the synaptic pole of the sensory cells, can act as an antagonist of the vestibular afferent transmitter at the postsynaptic level.

Effects of caloric stimuli on frog ampullar receptors.

The observation that caloric nystagmus can be evoked even in microgravity conditions argues against Barany's convective theory. To justify this result, gravity-independent mechanisms (mainly endolymphatic volume changes and direct action of the temperature on vestibular sensors) are believed to contribute to caloric-induced activation of vestibular receptors. To define the importance of both gravity-dependent and gravity-independent mechanisms, the posterior semicircular canal of the frog was thermally stimulated by a microthermistor positioned close to the sensory organ. The stimulus produced a gravity-dependent transcupular pressure difference that, depending on the position of the heater, could result in either excitation or inhibition of ampullar receptor sensory discharge. When the heater was positioned on the ampulla, or when the canal rested on the horizontal plane, no responses could be evoked by thermal stimuli. These results suggest that, in our experimental conditions (DeltaT up to 1.5 degrees C), neither a thermally induced expansion of the endolymph nor a direct action of the temperature on vestibular sensors play any major role.

The metabotropic glutamate receptors of the vestibular organs.

This research sought to test the presence and function of metabotropic excitatory amino acid receptors (mGluR) in the frog semicircular canal (SCC). The mGluR agonist +/- 1-aminocyclopentane-trans-1,3-dicarboxylate (ACPD) produced an increase in afferent firing rates of the ampullar nerve of the intact posterior canal. This increase was not due to a stimulation of cholinergic efferent terminals or the acetylcholine (ACh) receptor, since atropine, in concentrations which blocked the response to exogenous acetylcholine, did not affect the response to ACPD. Likewise, ACPD effects were not due to stimulation of postsynaptic NMDA receptors, since the NMDA antagonist D(-)-2-amino-5-phosphonopentanoate (AP-5) did not affect the response to ACPD, reinforcing the reported selectivity of ACPD for mGluRs. When the SCC was superfused with artificial perilymph known to inhibit hair cell transmitter release (i.e. low Ca-high Mg), ACPD failed to increase afferent firing. This suggests that the receptor activated by ACPD is located on the hair cell. Pharmacological evidence suggested that the mGluRs involved in afferent facilitation belong to Group I (i.e. subtypes 1 and 5). In fact, the Group III agonist AP-4 had no effect, and the ACPD facilitatory effect was blocked by the Group I mGluR antagonists (S)-4-carboxyphenylglycine (CPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Additional pharmacological evidence supported the presence of Group I mGluRs. Interestingly, the mGluR antagonists, AIDA and 4CPG, by themselves did not affect the resting firing rates of ampullar afferents. This may suggest that the mGluRs are not involved in resting activity but perhaps only in evoked activity (as suggested in Guth et al. (1991) Hear. Res. 56, 69-78). In addition, the mRNA for the mGluR1 has been detected in hair cells of both SCC, utricle, and saccule. In summary, the evidence points to an mGluR localized to the hair cell (i.e. an autoreceptor) which may be activated to produce a positive feedback augmentation of evoked but not resting transmitter release and thus affect afferent activity.

Regional distribution of calcium currents in frog semicircular canal hair cells.

In the present work we studied the regional expression of voltage-dependent Ca channels in hair cells from the frog semicircular canals, employing whole-cell patch-clamp on isolated and in situ hair cells. Although Ca channels are thought to play a major role in afferent transmission, up to now no data were available regarding their distribution in vestibular organs. The problem appears of interest, especially in the light of recent results showing the presence of multiple Ca current components in semicircular canal hair cells. Our data suggest the presence, in all regions of the crista ampullaris, of two classes of cells, one displaying an inactivating Ca current (R1) and one lacking it. In the former cells, Ca current amplitude decreased from the central to the peripheral zone (the maximal currents being observed in the intermediate zone). Only L-type and R2 current components displayed regional differences in expression, whereas the size and properties of R1, although variable among cells, were not regionalized. However, in cells lacking R1, Ca current amplitudes were similar regardless of cell shape and location. The possible contributions of this Ca current distribution to afferent discharge properties are discussed.

Experimental skeletal muscle revascularization by jejunal loop implant.

The possible onset of a vascular connection between the parietal network of a jejunal loop transposed into the crural region and the quadriceps muscle in hypoperfused condition has been experimentally studied. Ten dogs were included in this study. Sixty days after the operation the angiograms obtained by cannulating the artery of the implanted loop showed a rich neovascularity in the muscular area near the implant and the direct visualization of the venous outflow of the thigh. Histology of the specimens showed no substantial abnormalities of the muscular tissue. From these data the possible effective role of the skeletal muscle revascularization in the management of some severe ischemic conditions, after the failure of all the conventional reconstructive procedures, is suggested.

The origin of slow potentials in semicircular canals of the frog.

Slow potentials generated in the sensory organ of the ampulla were recorded in isolated semicircular canals of the frog by means of fluid electrodes. These potentials, which may be picked up from the intrampullar fluid and those from the ampullar nerve appear to be generated at different stages of the process taking place in crista ampullaris. Slow intra-ampullar potentials apparently reflect receptor potentials of hair cells. They are preserved after degeneration of the nerve fibre endings and are relatively insensitive to DNP poisoning; their amplitude is maximum at high K+ concentrations. Slow nerve potentials appear to be due to electronic spreading of post-synaptic excitatory potentials generated at cytoneural junctions. They disappear after degeneration of the nerve fibres, in low Ca++ and high Mg++ solutions and are extremely sensitive to DNP poisoning. An analysis of the time-course of the slow ampullar and nerve potentials referred to the discharge of impulses in afferent fibres was performed with a view to interpreting the transduction mechanism of semicircular canals.

Local mechanisms in vestibular receptor control. Effects of curare on the EPSPs and spike discharge recorded from single afferent fibres of the posterior canal nerve of the frog.

In the frog, the afferent discharge of EPSPs and spikes has been recorded intracellularly from single fibres of the posterior ampullar nerve before and after administration of D-tubocurarine (5 X 10(-6) M) in the following experimental conditions: (a) in animals in which the labyrinth was merely exposed (intact frogs), (b) in isolated acute labyrinth preparations, (c) in isolated chronic labyrinth preparations after degeneration of the efferent fibres. In both (a) and (c), curare induced either facilitation or inhibition of the resting discharge in a large number of units. By contrast, after degeneration of the efferent fibres, the effects of the drug were almost completely suppressed. These results indicate the existence of peripheral mechanisms involving the efferent nerve terminals which can control the release of afferent transmitter even in the absence of any central influence.

Ionic composition of the endolymph and sensory transduction in labyrinthine organs.

The role of various ions in stimuli conversion has been investigated in isolated frog semicircular canals, by replacing the endolymph with solutions deprived in turn of Na+, K+, Cl- and Ca++. Mechanical and electrical stimuli were employed and slow ampullar and nerve potentials were recorded, together with the afferent discharge of impulses in the eighth nerve. The results support evidence that K+ is the carrier of the receptor current, as postulated in Davis' hypothesis, while Ca++ is essential in the processes which allow the current to be modulated during cupula deflections and therefore endon hair cells with mechanical sensitivity.

The importance of potassium in the function of frog semicircular canals.

The slow potentials and afferent discharge of impulses in frog semicircular canals have been studied at different endolymphatic and perilymphatic K+ concentrations. Results indicate that the presence of K ions in the bathing fluids is essential for maintaining the receptor function in crista ampullaris, although very low concentrations of this ion in the perilymph are sufficient to preserve the receptor responsiveness to mechanical stimuli. The hypothesis is put forward that K+ may be pumped from the exterior of the canal towards the intracupular structures, where it accumulates. A K-rich endolymphatic environment does, however, appear to be necessary to ensure the resting activity of ampullar receptors and their ability to be "disfacilitated" during inhibitory cupula deflections.