Sikha Saha

Differential involvement of oriens/pyramidale interneurones in hippocampal network oscillations in vitro

Using whole‐cell patch‐clamp recordings in conjunction with post hoc anatomy we investigated the physiological properties of hippocampal stratum oriens and stratum pyramidale inhibitory interneurones, before and following the induction of pharmacologically evoked gamma frequency network oscillations. Prior to kainate‐induced transient epochs of gamma activity, two distinct classes of oriens interneurones, oriens lacunosum‐moleculare (O‐LM) and trilaminar cells, showed prominent differences in their membrane and firing properties, as well as in the amplitude and kinetics of their excitatory postsynaptic events. In the active network both types of neurone received a phasic barrage of gamma frequency excitatory inputs but, due to their differential functional integration, showed clear differences in their output patterns. While O‐LM cells fired intermittently at theta frequency, trilaminar interneurones discharged on every gamma cycle and showed a propensity to fire spike doublets. Two other classes of fast spiking interneurones, perisomatic targeting basket and bistratified cells, in the active network discharged predominantly single action potentials on every gamma cycle. Thus, within a locally excited network, O‐LM cells are likely to provide a theta‐frequency patterned output to distal dendritic segments, whereas basket and bistratified cells are involved in the generation of locally synchronous gamma band oscillations. The anatomy and output profile of trilaminar cells suggest they are involved in the projection of locally generated gamma rhythms to distal sites. Therefore a division of labour appears to exist whereby different frequencies and spatiotemporal properties of hippocampal rhythms are mediated by different interneurone subtypes.

A GABAergic projection from the central nucleus of the amygdala to the nucleus of the solitary tract: a combined anterograde tracing and electron microscopic immunohistochemical study

The central nucleus of the amygdala is involved in the modulation of autonomic, somatic and endocrine functions, as well as behavioural responses to stressful stimuli. Anatomical and physiological studies have suggested that this nucleus sends projections to the nucleus of the solitary tract, the primary site of termination of vagal and glossopharyngeal afferent fibres in the brain stem. To determine the neurochemical nature of the amygdaloid input to the nucleus of the solitary tract, anterograde tracing with biotinylated dextran amine was combined with post-embedding immunogold labelling for GABA and glutamate immunoreactivities and with pre-embedding labelling for the vesicular GABA transporter. Following injection of biotin dextran amine into the central nucleus of the amygdala, anterogradely labelled axons and varicosities were found throughout the rostrocaudal extent of the nucleus of the solitary tract, particularly in the medial, ventral and ventrolateral subnuclei. The anterogradely labelled terminals were found to make predominantly symmetrical synaptic contacts with dendrites, and occasionally onto cell bodies and dendritic spines, and to contain immunoreactivity for GABA and for the vesicular GABA transporter. Immunolabelling of serial sections with antibodies to glutamate showed that none of these axon terminals contained high enough densities of gold particle labelling to suggest that they contained other than low metabolic levels of glutamate immunoreactivity. These results provide conclusive evidence for a GABAergic pathway from the central nucleus of the amygdala to the nucleus of the solitary tract. This GABAergic projection may provide a substrate for inhibition of lower brain stem visceral reflexes, including baroreflex inhibition, through which the central nucleus of the amygdala could participate in cardiovascular regulation related to emotional behaviour and the defence reaction.

ROLE OF THE CENTRAL NUCLEUS OF THE AMYGDALA IN THE CONTROL OF BLOOD PRESSURE: DESCENDING PATHWAYS TO MEDULLARY CARDIOVASCULAR NUCLEI

1. One of the key areas that links psychologically induced stress with the blood pressure‐regulatory system is the central nucleus of the amygdala (CeA). This is an integratory forebrain nucleus that receives input from higher centres in the forebrain and has extensive connections with the hypothalamus and the medulla oblongata, areas involved in the regulation of the cardiovascular reflexes.

Glutamate, γ-aminobutyric acid and tachykin-inimmunoreactive synapses in the cat nucleus tractus solitarii

SummaryNeurophysiological and pharmacological evidence suggests that glutamate, γ-aminobutyric acid and tachykinins (substance P and neurokinin A) each have a role in cardiovascular regulation in the nucleus tractus solitarii. This study describes the ultrastructural relationships between nerve terminals immunoreactive for these substances in the nucleus tractus solitarii of the cat using post-embedding immunogold (single and double) labelling techniques on sections of tissue embedded in LR White resin. The technique combines a high specificity of labelling with good ultrastructural and antigenic preservation. Glutamate-immunoreactive terminals, recognized by their high density of gold particle labelling compared to the mean tissue level of labelling, accounted for about 40% of all synaptic terminals in the region of the nucleus tractus solitarii analysed (medial, dorsal, interstitial, gelatinosus and dorsolateral subnuclei). They appeared to comprise several morphological types, but formed mainly asymmetrical synapses, most often with dendrites of varying size, and contained spherical clear vesicles together with fewer dense-cored vesicles. Substance P- and neurokinin A-immunoreactive terminals were fewer in number (9% of all terminals) but similar in appearance, with the immunoreaction restricted to the dense-cored vesicles. Analysis of serial- and double-labelled sections showed a co-existence of substance P and neurokinin A-immunoreactivity in 21% of glutamate-immunoreactive terminals. Immunoreactivity for γ-aminobutyric acid was found in 33% of all terminals in the nucleus tractus solitarii. These predominantly contained pleomorphic vesicles and formed symmetrical synapses on dendrites and somata. Possible sites of axo-axonic contact by γ-aminobutyric acid-immunoreactive terminals onto glutamateor tachykinin-immunoreactive terminals were rare, but examples of adjacent glutamate and γ-aminobutyric acid-immunoreactive terminals synapsing on the same dendritic profile were frequent. These results provide an anatomical basis for a γ-aminobutyric acid mediated inhibition of glutamatergic excitatory inputs to the nucleus tractus solitarii at a post-synaptic level.

Glutamate‐immunoreactivity in identified vagal afferent terminals of the cat: a study combining horseradish peroxidase tracing and postembedding electron microscopic immunogold staining

Using electron microscopic immunohistochemistry we have shown that strong glutamate‐immunoreactivity (glutamate‐ir) is present in neuronal cell bodies of the nodose ganglion, axons in the tractus solitarius and afferent terminals in the nucleus tractus solitarii. Vagal afferent fibres were specifically labelled by transganglionic retrograde transport of horseradish peroxidase (HRP). Fifty‐seven per cent of the HRP‐labelled terminals in the dorsomedial medulla were found to contain a high level of glutamate‐ir, suggesting that a population of vagal afferent fibres uses glutamate as a neurotransmitter substance. There were no apparent ultrastructural differences between glutamate‐ir and non‐glutamate‐ir vagal afferent terminals, both classes mainly containing rounded vesicles and forming asymmetric synapses. However, some difference in their preference for postsynaptic target was noted. The great majority (83%) of non‐glutamate‐ir vagal afferent terminals made axodendritic synapses, but only just over half (57%) of the glutamate‐ir vagal terminals made synaptic contact with dendrites. Approximately 13% of the HRP‐labelled terminals were found to make synaptic contact with HRP‐labelled dendrites or soma of motoneurones of the dorsal vagal motor nucleus, confirming the existence of monosynaptic connections between vagal afferent fibres and vagal motoneurones.

Quantitative analysis and postsynaptic targets of GABA-immunoreactive boutons within the rat trigeminal motor nucleus

We have used the post-embedding immunogold labelling method using antibodies to gamma-aminobutyric acid (GABA) to obtain quantitative data on the distribution, frequency, postsynaptic targets and ultrastructural characteristics of GABA-immunoreactive (GABA-IR) boutons in the trigeminal motor nucleus of rats. We have also combined this method with horseradish peroxidase tracing to obtain specific evidence for termination of some GABA-IR boutons onto identified jaw-elevator motoneurones. Twenty-eight percent of all synapses in the motor nucleus involved GABA-IR boutons. Seventy-three percent of the GABA-IR boutons formed axo-dendritic synapses, 13% axo-somatic synapses and 14% axo-axonic synapses. Ninety-three percent of GABA-IR boutons formed symmetrical synapses. Overall, 58% of all boutons contained only flattened vesicles, while 26% contained round vesicles and 16% a mixture of vesicle types. Measurements of bouton cross sectional area, apposition length, and active zone length were obtained from serial reconstructions of 15 GABA-IR boutons and 30 unlabelled boutons. In each case mean values for GABA-IR boutons were significantly smaller than those for nonlabelled boutons.

γ-Aminobutyric Acid Receptor (GABAA) Subunits in Rat Nucleus Tractus Solitarii (NTS) Revealed by Polymerase Chain Reaction (PCR) and Immunohistochemistry

Expression of mRNAs encoding seven GABA(A) receptor subunits (alpha1, alpha2, alpha3, alpha5, beta2, beta3, gamma2) in the nucleus tractus solitarii (NTS) of rat medulla oblongata was examined by reverse transcription-polymerase chain reaction (RT-PCR). All subunit mRNAs, except alpha5, were clearly detected. Band densities produced by alpha1, alpha3, beta3, and gamma2 subunits were greater than those corresponding to beta2 and alpha2 transcripts. The localization of these subunits in tissue sections through NTS was examined by immunohistochemistry. The differential patterns of immunoreactivity in neuronal somata and dendrites of NTS neurons were generally in agreement with the PCR results, confirming that mRNA expression is correlated with receptor protein synthesis. At ultrastructural level, alpha1, alpha3, beta2/3, and gamma2 subunits were localized in both cytoplasmic and subsynaptic sites, the latter often apposed to GABA immunoreactive synapses. These results suggest that ionotropic receptors comprising the alpha1, alpha3, beta2/3, and gamma2 may mediate inhibitory GABA responses in the NTS.

Evidence for peptide co-transmission in retrograde- and anterograde-labelled central nucleus of amygdala neurones projecting to NTS

Synaptic terminals in the nucleus of the solitary tract (NTS) from axons originating in the central nucleus of the amygdala (CeA) are known to contain gamma-aminobutyric acid (GABA) immunoreactivity. Here, we have investigated whether such projections contain neuropeptides as putative co-transmitters. Somata in the medial and lateral CeA that were retrogradely labelled with cholera toxin B (CTb) injected into the commissural NTS were found to be immunoreactive for GABA, somatostatin (SOM), neurotensin (NT), vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS). Subpopulations of fibres in the NTS that were anterogradely labelled with biotin dextran amine (BDA) injected into the CeA and examined using both fluorescence and electron microscopy appeared to colocalise somatostatin, but not other neuropeptides. Their varicosities were observed in proximity to NTS neurones that were immunoreactive for the somatostatin receptor sst2A subtype, substance P (SP) NK1 receptor, and the GABAA receptor alpha3, beta1 and gamma2 subunits. This morphological evidence is consistent with the possibility of GABA-somatostatin co-transmission at synapses of some of the CeA projection neurones to NTS that might inhibit cardiovascular reflex responses in response to fear or emotion-related stimuli.

Central nucleus of amygdala projections to rostral ventrolateral medulla neurones activated by decreased blood pressure.

The central nucleus of amygdala (CeA) participates in cardiovascular regulation during emotional behaviour but it has not been established whether any of these effects are mediated through its direct connections to blood pressure‐regulating neurones in the rostral ventrolateral medulla (RVLM). The RVLM contains barosensitive neurones that maintain resting blood pressure via their projections to sympathetic preganglionic neurones in the thoracic spinal cord. In this study on rats, we used combined anterograde neuronal tracing of CeA projections with confocal and electron microscopic immunohistochemical detection of phenylethanolamine‐N‐methyltransferase, the adrenaline‐synthesizing enzyme present in C1 catecholamine neurones of the RVLM, and Fos, the protein product of the c‐fos proto‐oncogene. Fos expression in barosensitive neurones was stimulated by an intravenous infusion of the hypotensive agent sodium nitroprusside. Injection of the tracer biotin dextran amine (10‐kDa form) into the CeA resulted in anterograde labelling of axons and varicosities throughout the RVLM without retrograde labelling of somata in any brain area. With confocal microscopy, presumptive CeA terminals were found in close apposition to adrenergic (phenylethanolamine‐N‐methyltransferase‐immunoreactive) and non‐adrenergic neurones that displayed Fos‐immunoreactive nuclei in response to decreased blood pressure. Electron microscopic analysis confirmed that some labelled terminals of CeA axons made synaptic contact with c‐fos‐activated adrenergic neurones. The results provide evidence that cardiovascular influences elicited from the CeA during stressful events may be mediated, at least in part, via monosynaptic neural projections to barosensitive sympathetic blood pressure‐regulating neurones in the RVLM.

Light and electron microscopical localisation of 5-HT-immunoreactive boutons in the rat trigeminal motor nucleus

We have used pre-embedding EM immunohistochemical methods to obtain quantitative data on the frequency and post-synaptic targets of 5-hydroxytryptamine-immunoreactive (5-HT-IR) boutons within the rat V motor nucleus. Thirteen percent (69/531) of all synaptic contacts in the motor nucleus involved 5-HT-IR boutons. Seventy-four percent of 5-HT-IR boutons made axo-dendritic contacts, 20% axo-somatic contacts, and 6% axo-axonic contacts. We conclude that a significant fraction of boutons in the motor nucleus are 5-HT-IR and most contribute to postsynaptic rather than presynaptic effects on trigeminal motoneurones.